Immunomodulatory compounds and treatment of diseases related to an overproduction of inflammatory cytokines

ABSTRACT

Method of using immunomodulatory compounds for treating diseases related to an overproduction of inflammatory cytokines, including diseases selected from asthma, atopic dermatitis, allergic rhinitis, prostatitis, inflammatory bowel disease, diabetes, and rhumatoid arthritis, the compounds being of general formula (I): 
     
       
         
         
             
             
         
       
         
         
           
             wherein: 
             m and n, independently from each other, are an integer ranging from 1 to 4, 
             X and Y represent —COOH, —O—P(O)(OH) 2 , —O—SO 2 (OH), —NH 2 , —OH, —CONH(CH 2 ) n1 —NH 2 , —CO—NH—CH(COOH)—(CH 2 ) n1 —COOH, —CO—NH—CH(COOH)—(CH 2 ) n1 —NH 2 , —O—CO—(CH 2 ) n1 —NH 2 , —O—CO—(CH 2 ) n1 —CHOH—CH 2 OH, —O—CO—(CH 2 ) n1 —OH, —O—CO—(CH 2 ) n1 —COOH, —O—CO—(CH 2 ) n1 —CHO, —O—CO—(CH 2 ) n1 —NH—CO—(CH 2 ) n2 —COOH, 
             R 1  and R 2  each designate an acyl group derived from a saturated or unsaturated, straight- or branched-chain carboxylic acid having from 2 to 18 carbon atoms, which is unsubstituted or bears one to three substituents selected among hydroxyl, dihydroxyphosphoryloxy, alkyl of 2 to 18 carbon atoms, alkoxy of 2 to 18 carbon atoms, acyloxy of 2 to 18 carbon atoms in the acyl moiety, amino, acylamino.

The invention relates to the use of immunomodulatory compounds for thetreatment of diseases related to an overproduction of inflammatorycytokines, such as diseases selected from the group consisting ofasthma, atopic dermatitis, allergic rhinitis, prostatitis, inflammatorybowel disease, diabetes, and rhumatoid arthritis.

There have recently been major advances in our understanding of themechanism of activation of the innate immune system by microbialsignals. It has also become clear that such activation of the innateimmune system is essential for initiating adaptive immune responses andfor determining the type of adaptive responses that are generated.Together these observations have generated a huge renewal of interest inrational approaches to the development of immunotherapy.

Two major discoveries have been crucial to the increased understandingof innate immune activation:

-   1) the role of dendritic cells (DC) as the antigen-presenting cells    responsible for the induction of primary immune responses,-   2) the discovery of Toll-like receptors (TLR) and other “pattern    recognition” receptors that detect the presence of microbial    structures, leading to the stimulation of the innate immune system.

Sallusto and Lanzavecchia first reported that immature DC can begenerated from human monocytes in medium supplemented with GM-CSF andIL-4 (Sallusto F, Lanzavecchia A. J Exp Med 1994, 179:1109-18). However,it rapidly became apparent that, in addition to GM-CSF and IL-4, afurther maturation factor was required to obtain mature DC that werefully functional as antigen-presenting cells (APC). Mature DC arecapable of initiating and steering the adaptive immune response.

Such maturation signals can be provided by microbial products. Theseproducts have been show to act through TLRs. Eleven TLRs have beenidentified, capable or recognising different microbial products. TLR4 isthe receptor that specifically recognises bacterial lipopolysaccharide(LPS), one of the most potent stimulators of the innate immune systemknown.

International application WO 2005 007699 describes the use of specificbinding members, in particular of human anti-IL-13 antibody moleculesand especially those which neutralise IL-13 activity, in the diagnosisor treatment of IL-13 related disorders, including asthma, atopicdermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease andHodgkin's lymphoma.

However investigations are still necessary to know the exact role ofIL-13 in immunomodulation and the interpretation of the results relatedto the study of the IL-13/IL-4 pathway are still of hypothetical nature,as shown by the following two recent publications.

The publication “IL-4/IL-13 pathway genetics strongly influence serumIgE levels and childhood asthma” by Michael Kabesch et al., (J. AllergyClin. Immunol. Volume 117, Number 2) states that IgE production, ahallmark of asthma and atopic disease, may be under genetic controlinvolving genes of the IL-4 and IL-13 pathway. Because IgE switching isfundamental for human immunity and survival, the regulation of IgE isdelicate. Although the IL-4/IL-13 pathway may influence the developmentof asthma mainly through its effect on IgE regulation, the results givenindicate that this may not exclusively be the case.

The publication “Control of allergic airway inflammation throughimmunomodulation” by David B Corry, and Farrah Kheradmand, (J. AllergyClin. Immunol. Volume 117, Number 2), describes the major findingslinked with clinical trials which have changed the management of asthma,and which have not, and that deserve further study. It is reported thatIL-4, a cytokine that is required for B-cell IgE responses, is criticalto type I hypersensitivity responses, and that IL-4 was therefore one ofthe first cytokines to be targeted in asthma clinical trials by using asoluble form of the IL-4 receptor α chain (sIL-4Rα), which binds to andinactivates IL-4. IL-13 is another cytokine that has many of the sameeffects as IL-4 but is not inhibited by sIL-4α. The conclusion is thatfuture studies might therefore attempt to inhibit both IL-4 and IL-13simultaneously for the treatment of asthma.

The present invention relies on the demonstration by the inventors thatthe molecules as described hereafter are efficient agonists of TLR4 witha good safety profile, that are able to induce the complete maturationof functional monocyte-derived DC, as well activating other cell-typesexpressing TLR4. DC's treated with compounds according to the inventionare able to induce primary T-cell activation and to favour Th1 cellularimmune responses.

The inventors also demonstrated that compounds according to theinvention are able to markedly decrease the secretion of IL-13 by CD4⁺ Tcells polyclonally-activated by anti-CD3 and anti-CD28 antibodies.

Moreover, when one of the compounds was administered by the i.p. or i.n.routes to a murine model of asthma, the inventors demonstrate aprevention of the allergic responses produced by antigen sensitisationand subsequent aerosol exposure to antigen. The immunological parametersmeasured suggest a relative decrease in Th2 responses, which may explainthe mechanism by which compounds according to the invention are actingin this asthma model. Additionally, the authors demonstrate that thecompound of the invention is able to decrease diabetes occurrence in NonObese Diabetic mice.

The present invention relates to a method for treating warm-bloodedanimals including humans, suffering from a disease or disorder relatedto an overproduction of inflammatory cytokines, comprising theadministration to a patient in need thereof of an appropriate amount ofa pharmaceutical composition comprising at least one immunomodulatorycompound of the following general formula (I):

Wherein

-   -   m and n, independently from each other, are an integer ranging        from 1 to 4,    -   X and Y each designate a group either in neutral or charged        state, selected from the following groups:        -   carboxyl —COOH,        -   dihydroxyphosphoryloxy —O—P(O)(OH)₂,        -   hydroxysulfonyloxy —O—SO₂(OH),        -   amino —NH₂,        -   hydroxyl —OH,        -   [amino(C₁-C₁₀)alkyl]aminocarbonyl —CONH(CH₂)_(n1)—NH₂, with            n₁ being an integer from 1 to 10,        -   [dicarboxy(C₁-C₅)alkyl]aminocarbonyl            —CO—NH—CH(COOH)—(CH₂)_(n1)—COOH, with n₁ being an integer            from 1 to 5,        -   {carboxy[amino(C₁-C₅)alkyl]}aminocarbonyl            —CO—NH—CH(COOH)—(CH₂)_(n1)—NH₂, with n₁ being an integer            from 1 to 5,        -   amino(C₁-C₁₅)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being            an integer from 1 to 15,        -   dihydroxy(C₁-C₁₀)alkanoyloxy —O—CO—(CH₂)₁—CHOH—CH₂OH, with            n, being an integer from 1 to 10,        -   hydroxy(C₁-C₁₀)alkanoyloxy —O—CO—(CH₂)_(n1)—OH, with n₁            being an integer from 1 to 10,        -   carboxy(C₁-C₁₀)alkanoyloxy —O—CO—(CH₂)_(n1)—COOH, with n₁            being an integer from 1 to 10,        -   oxo(C₁-C₅)alkanoyloxy —O—CO—(CH₂)_(n1)—CHO, with n₁ being an            integer from 1 to 5,        -   [carboxy(C₁-C₁₀)alkanoyl]amino(C₁-C₁₅)alkanoyloxy            —O—CO—(CH₂)_(n1)—NH—CO—(CH₂)_(n2)—COOH, with n₁ being an            integer from 1 to 10, and n₂ being an integer from 1 to 15,    -   R₁ and R₂ each designate an acyl group derived from a saturated        or unsaturated, straight- or branched-chain carboxylic acid        having from 2 to 18 carbon atoms, which is unsubstituted or        bears one to three substituents selected among hydroxyl,        dihydroxyphosphoryloxy, alkyl of 2 to 18 carbon atoms, alkoxy of        2 to 18 carbon atoms, acyloxy of 2 to 18 carbon atoms in the        acyl moiety, amino, acylamino,    -   C^(*1) and C^(*2), independently from each other being asymetric        carbons in configuration R, S, or in the racemic form RS,        or a pharmaceutically acceptable salt, solvate or isomer        thereof, optionally in conjugation or admixture with an inert        non-toxic pharmaceutically acceptable diluent or carrier.

The invention relates more particularly to a method as defined above,wherein

-   -   X is selected from the following groups:        -   carboxyl —COOH,        -   dihydroxyphosphoryloxy —O—P(O)(OH)₂,        -   hydroxysulfonyloxy —O—SO₂(OH),        -   amino —NH₂,        -   [amino(C₁-C₁₀)alkyl]aminocarbonyl —CONH(CH₂)_(n1)—NH₂, with            n₁ being an integer from 1 to 10,        -   [dicarboxy(C₁-C₅)alkyl]aminocarbonyl            —CO—NH—CH(COOH)—(CH₂)_(n1)—COOH, with n₁ being an integer            from 1 to 5,        -   {carboxy[amino(C₁-C₅)alkyl]}aminocarbonyl            —CO—NH—CH(COOH)—(CH₂)_(n1)—NH₂, with n₁ being an integer            from 1 to 5,        -   amino(C₁-C₁₅)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being            an integer from 1 to 15,    -   and Y is selected from the following groups:        -   dihydroxyphosphoryloxy —O—P(O)(OH)₂,        -   hydroxysulfonyloxy —O—SO₂(0H),        -   amino —NH₂,        -   hydroxyl —OH,        -   amino(C₁-C₁₅)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being            an integer from 1 to 15,        -   dihydroxy(C₁-C₁₀)alkanoyloxy —O—CO—(CH₂)_(n1)—CHOH—CH₂OH,            with n₁ being an integer from 1 to 10,        -   hydroxy(C₁-C₁₀)alkanoyloxy —O—CO—(CH₂)_(n1)—OH, with n₁            being an integer from 1 to 10,        -   carboxy(C₁-C₁₀)alkanoyloxy —O—CO(CH₂)_(n1)—COOH, with n₁            being an integer from 1 to 10,        -   oxo(C₁-C₅)alkanoyloxy —O—CO—(CH₂)_(n1)—CHO, with n₁ being an            integer from 1 to 5,        -   [carboxy(C₁-C₁₀)alkanoyl]amino(C₁-C₁₅)alkanoyloxy            —O—CO—(CH₂)_(n1)—NH—CO—(CH₂)_(n2)—COOH, with n₁ being an            integer from 1 to 10, and n₂ being an integer from 1 to 15.

The invention concerns more particularly a method as defined above,wherein

-   -   X is selected from the following groups:        -   carboxyl —COOH,        -   dihydroxyphosphoryloxy —O—P(O)(OH)₂,        -   hydroxysulfonyloxy —O—SO₂(OH),        -   amino —NH₂,        -   [amino(C₁-C₆)alkyl]aminocarbony)-CONH(CH₂)_(n1)—NH₂, with n₁            being an integer from 1 to 6,        -   [dicarboxy(C₁-C₅)alkyl]aminocarbonyl            —CO—NH—CH(COOH)—(CH₂)_(n1)—COOH, with n, being an integer            from 1 to 5,        -   {carboxy[amino(C₁-C₅)alkyl]}aminocarbonyl            —CO—NH—CH(COOH)—(CH₂)_(n1)—NH₂, with n₁ being an integer            from 1 to 5,        -   amino(C₂-C₁₂)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being            an integer from 2 to 12,    -   and Y is selected from the following groups:        -   dihydroxyphosphoryloxy —O—P(O)(OH)₂)        -   hydroxysulfonyloxy —O—SO₂(OH),        -   amino —NH₂,        -   hydroxyl —OH,        -   amino(C₂-C₁₂)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being            an integer from 2 to 12,        -   dihydroxy(C₃-C₇)alkanoyloxy —O—CO—(CH₂)_(n1)—CHOH—CH₂OH,            with n₁ being an integer from 3 to 7,        -   hydroxy(C₂-C₆)alkanoyloxy —O—CO—(CH₂)_(n1)—OH, with n₁ being            an integer from 2 to 6,        -   carboxy(C₃-C₆)alkanoyloxy —O—CO—(CH₂)_(n1)—COOH, with n₁            being an integer from 3 to 6,        -   oxo(C₂-C₅)alkanoyloxy —O—CO—(CH₂)_(n1)—CHO, with n₁ being an            integer from 2 to 5,        -   [carboxy(C₃-C₆)alkanoyl]amino(C₂-C₁₂)alkanoyloxy            —O—CO—(CH₂)_(n1)—NH—CO—(CH₂)_(n2)—COOH, with n₁ being an            integer from 3 to 6, and n₂ being an integer from 2 to 12.

The invention relates more particularly to a method as defined above,wherein

-   -   X is selected from the following groups:        -   —COOH,        -   —O—P(O)(OH)₂,        -   —O—SO₂(OH),        -   —NH₂,        -   —CO—NH—(CH₂)₃—NH₂, or —CONH—(CH₂)₆—NH₂,        -   —CO—NH—CH(COOH)—CH₂—COOH,        -   —CO—NH—CH(COOH)—(CH₂)₄—NH₂,        -   —O—CO—(CH₂)₅—NH₂,    -   and Y is selected from the following groups:        -   —O—P(O)(OH)₂,        -   —O—SO₂(OH),        -   —NH₂,        -   —OH,        -   —O—CO—CH₂—NH₂ (2-aminoethanoyloxy), —O—CO—(CH₂)₂—NH₂            (3-aminopropanoyloxy), —O—CO—(CH₂)₅—NH₂            (6-aminohexanoyloxy), or —O—CO—(CH₂)₁₁—NH₂            (12-aminododecanoyloxy),        -   —O—CO—(CH₂)₄—CHOH—CH₂OH (6,7-dihydroxyheptanoyloxy),        -   —O—CO—(CH₂)₅—OH (6-hydroxyhexanoyloxy),        -   —O—CO—(CH₂)₂—COOH (3-carboxypropanoyloxy)        -   —O—CO—(CH₂)₄—CHO (6-oxohexanoyloxy),        -   —O—CO—(CH₂)₅—NH—CO—(CH₂)₂—COOH (3-carboxypropanoylamino            hexanoyloxy).

The invention concerns more particularly a method as defined above,wherein R₁ and R₂ are chosen among:

-   -   —CO—CH₂—C*H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃, (3(C₁₂O)C₁₄),    -   —CO—CH₂—C*HOH—(CH₂)₁₀—CH₃, (3(HO)C₁₄),    -   —CO—CH₃, (C2),    -   —CO—CH₂—NH—CO—C*H[O—CO—(CH₂)₈—CH₃]—(CH₂)₅—CH₃, ([2(C₁₀O)C₈]NC₂),    -   —CO—C*H[O—CO—(CH₂)₄—CH₃]-(CH₂)—CH₃, (2(C₆O)C₈),    -   —CO—(CH₂)₁₆—CH₃, (C18),    -   —CO—CH₂—C*H[O—CH₂—C₆H₅]—(CH₂)₁₀—CH₃, (3(BnO)C₁₄),    -   —CO—CH₂—C*H[O—P(O)(OH)₂]—(CH₂)₁₀—CH₃, (3[(OH)₂—P(O)O]C₁₄),

C* corresponding to an asymetric carbon in configuration R, S, or in theracemic form RS, in the formulae mentioned above.

The invention relates more particularly to a method as defined above,wherein

-   -   R₁ is chosen among:        -   —CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]-(CH₂)₁₀—CH₃,            (3(C₁₂O)C₁₄),        -   —CO—CH₂—C^(**1)HOH—(CH₂)₁₀—CH₃, (3(HO)C₁₄),        -   —CO—CH₃, (C2),        -   —CO—CH₂—NH—CO—C^(**1)H[O—CO—(CH₂)₈—CH₃]—(CH₂)₅—CH₃,            ([2(C₁₀O)C₈]NC₂),        -   —CO—C^(**1)H[O—CO—(CH₂)₄—CH₃]—(CH₂)₅—CH₃, (2(C₆O)C₈),        -   —CO—(CH₂)₁₆—CH₃, (C18),    -   R₂ is chosen among:        -   —CO—CH₂—C^(**2)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,            (3(C₁₂O)C₁₄),        -   —CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, (3(HO)C₁₄),        -   —CO—CH₂—C^(**2)H[O—CH₂—C₆H₅]—(CH₂)₁₀—CH₃, (3(BnO)C₁₄),        -   —CO—CH₂—C^(**2)H[O—P(O)(OH)₂]—(CH₂)₁₀—CH₃,            (3[(OH)₂—P(O)O]C₁₄).

C^(**1) and C^(**2) corresponding to asymetric carbons in configurationR, S, or in the racemic form RS, in the formulae mentioned above.

The invention also relates to a method as defined above, wherein theadministered compound has the general formula (II):

Wherein X, Y, R₁, R₂, and m are as defined above, C^(*1) is inconfiguration R, S, or is the racemic RS, and C^(*2) is in configurationR.

The invention relates more particularly to a method as defined above,wherein the administered compound is selected among those of formula(II) wherein:

-   -   X=—O—P(O)(OH)₂, Y=—O—P(O)(OH)₂,        R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,        R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=2, C^(*1) is in        configuration S, C^(*2) is in configuration R, C^(**1) and        C^(**2) are in configuration R, i.e.        (3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoyl        amino]-decan-1,10-diol (1,10)-bis-dihydrogenophosphate        (OM-294-DP (S,R)),    -   X=—O—P(O)(OH)₂, Y=—O—CO—(CH₂)₄—CHOH—CH₂OH,        R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,        R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=2, C^(*1) is in        configuration S, and C^(*2) is in configuration R, C^(**1) and        C^(**2) are in configuration R, i.e. (3S,        9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxy        tetradecanoyl amino]-decan-1, 10-diol 1-dihydrogenophosphate        10-(6,7-dihydroxyheptanoate) (OM-197-MP-HD (S,R)),    -   X=—COOH, Y=—O—CO—(CH₂)₄—CHOH—CH₂OH,        R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,        R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=1, C^(*1) is in        configuration S, and C^(*2) is in configuration R, C^(**1) and        C^(**2) are in configuration R, i.e.        N-[(R)-3-dodecanoyloxytetradecanoyl]-L-aspartic acid,        α-N-{(4R)-5-hydroxy-4-[(R)-3-hydroxytetradecanoylamino]pentyl}amide        5-O-(6,7-dihydroxyheptanoate) (OM-197-MC-HD (S,R)),    -   X=—O—P(O)(OH)₂, Y=—O—CO—(CH₂)₅—NH₂,        R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,        R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=2, C^(*1) is in        configuration S, and C^(*2) is in configuration R, C^(**1) and        C^(**2) are in configuration R, i.e.        (3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetra        decanoylamino]-decan-1,10-diol 1-dihydrogeno phosphate        10-(6-aminohexanoate) (OM-197-MP-AC(S,R)),    -   X=—NH₂, Y=—O—P(O)(OH)₂,        R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,        R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=4, C^(*1) is in        configuration S, and C^(*2) is in configuration R, C^(**1) and        C^(**2) are in configuration R, i.e.        (5S,11R)-1-Amino-5-[(R)-3-dodecanoyloxytetradecanoylamino]-6-oxo-7-aza-11-[(R)-3-hydroxytetra        decanoylamino]-dodecan-12-ol 12-dihydrogen phosphate        (OM-294-BA-MP (S,R)).

The invention also concerns a method as defined above, for treatingwarm-blooded animals including humans, suffering from a disease ordisorder related to an overproduction of inflammatory cytokines orinflammatory disease markers by activated T lymphocytes, monocytes, orantigen presenting cells in the organism, wherein the inflammatorycytokines or inflammatory disease markers belong to the group consistingof IL-1β, IL-4, IL-5 IL-6, IL-8, IL-9, IL-13, IFN-γ, TNF-α, and MCP-1.

The invention relates more particularly to a method as defined above,wherein the disease is selected from the group consisting of asthma,diabetes, atopic dermatitis, allergic rhinitis, prostatitis,inflammatory bowel disease, and rhumatoid arthritis.

The invention also relates more particularly to a method as definedabove, which consists in administering a therapeutically effectiveamount of any compound of formula (I) as defined above of the inventionin a pharmaceutically-acceptable carrier, excipient or formulation, viaa mucosal or parenteral route.

The invention also concerns more particularly a method as defined above,which consists in administering a therapeutically effective amount of acompound of formula (I) as defined above of the invention preferentiallyvia the peritoneal, subcutaneous, oral, intranasal, sublingual, oraerosol routes.

The invention relates more particularly to gastroresistantpharmaceutical compositions comprising a therapeutically effectiveamount of a compound of formula (I) as defined above in association witha gastroresistant carrier such as hydrophilic poloxamer surfactants suchas poloxamer 407 (Lutrol F-127). Colloidal carriers such as polymericnanoparticules or microparticules are also appropriate formulations forthe oral delivery of formula (I) when enteric polymers such asmethacrylate polymers are used.

In a more conventional way the use of gastroresistant tablets orcapsules obtained by the use of an enteric coating could also be analternative for the oral route.

The invention also relates more particularly to a method as definedabove, wherein the needed dosages of the immunomodulatory molecules offormula (I) as defined above of the invention range from 0.01 to 50mg/m² in humans.

The invention also relates to the use of at least one compound offormula (I) as defined above for the preparation of a drug for theprevention or treatment of a disease or disorder such as asthma,diabetes, atopic dermatitis, allergic rhinitis, prostatitis,inflammatory bowel disease, and rhumatoid arthritis, related to anoverproduction of inflammatory cytokines or inflammatory diseasemarkers.

The invention also concerns the compounds of formula (I) as definedabove, and the pharmaceutical containing said compounds in associationwith a physiologically acceptable carrier.

Compounds of formula (I) as defined above of the present invention canbe prepared according to the method described in WO 00/00462 and WO01/46127 relative to the preparation and the use of compounds of formula(I) in the treatment of cancers, or as immunoadjuvants.

The invention will be further described in details in the followingdescription of the synthesis of compounds OM-294-DP (S,R), OM-197-MP-HD(S,R), OM-197-MC-HD (S,R), OM-197-MP-AC (S,R), and OM-294-BA-MP (S,R),and of their biological properties.

EXAMPLES Example 1(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol1-dihydrogenophosphate 10-(6-aminohexanoate) (=OM-197-MP-AC (S,R))(Scheme 1) 1.(2R)-5-(Benzyloxycarbonylamino)-2-[(R)-3-benzyloxytetradecanoylamino]-1-(2-tetrahydropyranyloxy)pentane(C-1)

To a solution of(2R)-5-(benzyloxycarbonylamino)-2-[(R)-3-benzyloxytetra-decanoylamino]pentan-1-ol(PCT WO00146127A1) (2.5 g; 4.39 mmol) in anhydrous CH₂Cl₂ (83 ml) atroom temperature and under argon were added successively3,4-dihydro-2H-pyran (DHP) (1.4 ml, 15.38 mmol) then pyridiniump-toluenesulfonate (PPTS) (441 mg, 1.75 mmol). The solution was stirredfor 18 h at room temperature then diluted with CH₂Cl₂ (100 ml), washedwith 5% aqueous NaHCO₃ then with H₂O. The organic phase was dried overMgSO₄, filtered and concentrated. Purification by flash chromatographyon silica gel (AcpOEt/pet. ether 4/1) gave compound C-1 (2.86 g; 100%)as a white crystalline solid. (Rf=0.66 in AcOEt/pet. ether 4/1; U.V. andphosphomolybdate).

C₃₉H₆₀N₂O₆. IS/MS: m/z 653.5 ([M+H]⁺), 675.5 ([M+Na]⁺). Mp=84-86° C.

2.(2R)-5-Amino-2-[(R)-3-benzyloxytetradecanoylamino]-1-(2-tetrahydropyranyloxy)-pentane(C-2)

A solution of compound C-1 (2.5 g; 4.4 mmol) in EtOH (150 ml) containingtriethylamine (4 ml) was hydrogenated in the presence of 10% Pd on C atroom temperature and under atmospheric pressure of hydrogen for 3.5 h.The catalyst was then removed by filtration, washed with ethanol and thefiltrate was concentrated and dried under high vacuum to give the freeamine C-2 (2.15 g; 96%) as an amorphous, white solid. C₃₁H₅₄N₂O₄. IS/MS:m/z 519.5 ([M+H]⁺).

3. (S)-(α)-[(R)-3-Dodecanoyloxytetradecanoylamino]-γ-butyrolactone (C-3)

To a solution of (R)-3-dodecanoyloxytetradecanoic acid [Bull. Chem. Soc.Jpn 60 (1987), 2205-2214] (2.16 g; 5.1 mmol) in anhydrous THF (28 ml) at−15° C. and under argon were added successively N-methylmorpholine (0.56ml; 5.1 mmol; 1 eq) and isobutyl chloroformate (657 μl; 5.1 mmol; 1 eq).After 1 h under stirring at −15° C., L-homoserine lactone hydrobromide(916 mg; 5.1 mmol; 1 eq) as a solution in 0.72 M aqueous NaHCO₃ (14 ml,2 eq) was added. The reaction mixture was stirred for 20 h at roomtemperature. The mixture was diluted with Et₂O (130 mL), the organicphase was separated and washed with H₂O then dried over MgSO₄, filteredand concentrated. A purification by crystallization (minimum volume ofCH₂Cl₂ and excess pentane at 0° C.) gave compound C-3 (2.17 g; 85%) as awhite solid. C₃₀H₅₅NO₅. IS/MS: m/z 510.5 ([M+H]⁺), 532.5 ([M+Na]⁺).mp=79-80° C.

4.(3S-9R)-3-[(R)-3-Dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradecanoylamino]-10-(2-tetrahydropyranyl)oxy-decan-1-ol(C-4)

To a solution of compound C-2 (638 mg, 1.23 mmol, 1.3 eq) in anhydrousCH₂Cl₂ (1.5 mL) at 20-21° C. was added compound C-3 (483 mg, 0.95 mmol).The solution was stirred for 3 days at 20-21° C.; the solvent wasevaporated under reduced pressure. A purification by flashchromatography on silica gel (CH₂Cl₂/acetone 5/1 to 1/1) gave alcoholC-4 (829 mg, 85%) as a white solid. C₆₁H₁₀₉N₃O₉. IS/MS: m/z 1029.0([M+H]⁺), 1051.0 ([M+Na]⁺). mp=81-82° C.

5.(3S,9R)-3-[(R)-3-Dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradécanoylamino]-10-(2-tetrahydropyranyl)oxy-decan-1-oldibenzyl phosphate (C-5)

To a solution of alcohol C-4 (120 mg; 0.12 mmol) and 1H-tetrazole (25mg; 0.35 mmol; 3 eq) in anhydrous THF (5 ml) at room temperature andunder argon was added N,N-dibenzyl diethylphosphoramidite (85%, 95 μl;0.27 mmol; 2.3 eq). After 45 min under stirring, the reaction mixturewas cooled down to −40° C. then a solution of mCPBA (57-86%; 75 mg; 0.43mmol; 3.7 eq) in CH₂Cl₂ (3 ml) was added. After 45 min at −40° C., themixture was warmed up and a saturated solution of Na₂S₂O₃ (3 ml) wasadded and the mixture was stirred for 10 min. The solution was dilutedwith ether, the organic phase was separated and washed with saturatedNa₂S₂O₃ (5×), then with saturated NaHCO₃ (2×). The organic phase wasdried over MgSO₄, filtered and concentrated. Purification by flashchromatography on silica gel (CH₂Cl₂/acetone 4/1 then 2/1) gave dibenzylphosphate C-5 (126 mg; 84%) as an amorphous solid.

6.(3S,9R)-3-[(R)-3-Dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradecanoylamino]-decan-1,10-diol1-(dibenzyl phosphate) (C-6)

To a 1% HCl solution in methanol (25 ml) at 0° C. was added a solutionof compound C-5 (700 mg, 0.54 mmol) in CH₂Cl₂ (2.5 ml). After 45 minunder stirring at 0° C., the reaction mixture was neutralized with 5%aqueous NaHCO₃, diluted with CH₂Cl₂ then the organic phase wasseparated. The aqueous phase was extracted with CH₂Cl₂ (3×) then theorganic phases were combined, dried over MgSO₄, filtered andconcentrated to give alcohol C-6 (640 mg; 98%).

7.(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradecanoylamino]-decan-1,10-diol1-dibenzyl phosphate 10-(6-benzyloxycarbonylaminohexanoate (C-7)

To a solution of the compound prepared above C-6 (640 mg, 0.53 mmol.)and 6-(benzyloxycarbonylamino)hexanoic acid (423 mg, 1.60 mmol.) in dryCH₂Cl₂ (25 ml) at 0° C. and under argon flow, there are added insuccession commercially available1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (306 mg,1.60 mmol.) and 4-dimethylaminopyridine (20 mg, 160 μmol.). The reactionmixture is then stirred for 30 minutes at 0° C. and thereafter overnightat room temperature. The reaction medium is then washed with water and asolution of 1N HCl followed by layer separation. The organic layer isdried over MgSO₄, filtered and evaporated. By running a flashchromatography purification on a silica gel (4/1 then 2/1 CH₂Cl₂/acetoneeluent), there is recovered the coupling reaction product C-7 (537 mg;71%). ¹³C-NMR (62.89 MHz, CDCl₃), δ in ppm: 173.18, 171.16, 170.38,169.60, 156.30, 138.23, 136.50, 135.38, 135.28, 128.42, 128.26; 128.17,127.79, 127.74, 127.44, 76.48, 71.15, 70.84, 69.47, 69.39, 69.31, 66.25,65.62, 64.37, 49.78, 47.76, 41.41, 41.34, 40.57, 38.97, 34.22, 34.16,33.96, 33.57, 32.95, 31.70, 29.15, 28.95, 28.32, 25.87, 25.46, 25.02,28.80, 24.18, 22.49, 13.94.

8.(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol1-dihydrogenphosphate 10-(6-aminohexanoate) (C-8) (=OM-197-MP-AC (S,R))

A solution of the compound C-7 (500 mg, 0.35 mmol.) in a 5/2CH₂Cl₂/ethanol mixture (70 ml) containing acetic acid (10 ml) ishydrogenated in presence of Pd on carbon containing 10% Pd at roomtemperature and under atmospheric pressure hydrogen for 12 to 24 hours.The catalyst is filtered off. The filtrate is evaporated to dryness andthe residue is then dried by suction from a vacuum pump to obtain C-8(368 mg, quantitative yield). ES/MS: m/z ratio 1047.9 [M+H]⁺; 1069.8

Example 2(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol1-dihydrogenophosphate 10-(6,7-dihydroxyheptanoate) (=OM-197-MP-HD(S,R)) (Scheme 2) 1. Benzyl 6-heptenoate (C-9)

To a solution of 6-heptenoic acid (4.71 g, 36.75 mmol) in AcOEt (80 mL)at 0° C. were successively added triethylamine (15.3 mL, 110.24 mmol),benzyl bromide (13.1 mL, 110.24 mmol) and Bu₄NI (6.79 g, 18.37 mmol).The reaction mixture was stirred at 0° C. for 2H and concentrated invacuo. The residue is taken up in AcOEt, the organic phase was washedwith a saturated aqueous NaHCO₃ and H₂O. The organic phase was driedover MgSO₄ and the solvent removed in vacuo. Flash chromatography of theresidue on silica gel (n-heptane/EtOAc, 9:1) provided compound C-9 (6.00g 75%) as a colorless oil. ¹³C-NMR (62.89 MHz, CDCl₃): 24.42, 28.34,33.37, 34.13, 66.08, 114.73, 128.19, 128.55, 136.16, 138.37, 173.43.

2. Benzyl 6,7-epoxyheptanoate (C-10)

To a solution of m-CPBA (2.76 g, 12.29 mmol) in CH₂Cl₂ (50 mL) at 0° C.was slowly added a solution of C-9 (1.79 g, 8.19 mmol) in CH₂Cl₂ (20mL). The solution was stirred at 0° C. for 2H then at room temperaturefor 18 H. The solution was diluted with CH₂Cl₂ and the organic phase waswashed with a saturated aqueous Na₂S₂O₃ solution (5×). The organic phasewas dried over MgSO₄ and the solvent removed in vacuo. Flashchromatography of the residue on silica gel (n-heptane/EtOAc, 5:1)provided compound C-10 (1.54 g; 80%) as a colorless oil. ¹³C-NMR (62.89MHz, CDCl₃): 24.60, 25.39, 32.00, 34.02, 46.86, 51.91, 66.04, 128.11,128.46, 136.01, 173.17.

3. Benzyl 6,7-Isopropylideneheptanoate (C-11)

To a solution of C-10 (1.54 g, 6.59 mmol) in acetone (50 mL) at roomtemperature was added sulfuric acid (0.42 mL, 7.9 mmol). The solutionwas stirred for 3H, diluted with diethyl ether and the organic phasewashed with a saturated aqueous NaHCO₃ solution. The organic phase wasdried over MgSO₄ and the solvent removed in vacuo to give C-11 (1.73 g)as a pale yellow oil which was used in the next step without furtherpurification.

4. 6,7-Isopropylideneheptanoic acid (C-12)

A solution of the compound C-11 (1.53 g) in EtOAc (20 ml) containingEt₃N (3.65 mL, 26.16 mmol) was hydrogenated in presence of Pd on carboncontaining 10% Pd at room temperature and under atmospheric pressurehydrogen for 3 h. The catalyst is filtered off and the filtrate isevaporated to dryness. Flash chromatography of the residue on silica gel(CH₂Cl₂/MeOH, 5:1) provided compound C-12 (0.63 g; 54% over 2 steps) asa colorless oil.

5.(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradecanoylamino]-decan-1,10-diol1-dibenzyl phosphate 10-(6,7-isopropylideneheptanoate (C-13)

To a solution of the compound prepared above C-6 (248 mg, 0.21 mmol.)and compound 6,7-Isopropylideneheptanoic acid (C-12) (92 mg, 0.45 mmol.)in dry CH₂Cl₂ (5 ml) at 0° C. and under argon flow, there are added insuccession commercially available1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (91 mg, 0.47mmol.) and 4-dimethylaminopyridine (catalytic). The reaction mixture isthen stirred for 30 minutes at 0° C. and thereafter overnight at roomtemperature. The reaction medium is then washed with water and asolution of 1N HCl followed by layer separation. The organic layer isdried over MgSO₄, filtered and evaporated. By running a flashchromatography purification on a silica gel (CH₂Cl₂/MeOH, 99:1), thereis recovered the coupling reaction product C-13 (240 mg; 83%). ES/MS:m/z 1390 [M+H]⁺; 1411 [M+Na]⁺.

6.(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol1-dihydrogenphosphate 10-(6,7-dihydroxyheptanoate) (C-14) (=OM-197-MP-HD(S,R))

A solution of the compound C-13 (180 mg, 0.13 mmol.) in a 5/2CH₂Cl₂/ethanol mixture (21 ml) containing acetic acid (3 ml) ishydrogenated in presence of Pd on carbon containing 10% Pd at roomtemperature and under atmospheric pressure hydrogen for 12 to 36 hours.The catalyst is filtered off. The filtrate is evaporated to dryness andthe residue is then dried by suction from a vacuum pump to obtain C-14(139 mg, quantitative yield). ES/MS: m/z 1079 [M+H]⁺; 1101 [M+Na]⁺.

Example 3(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol1,10-bis-dihydrogenophosphate) (=OM-294-DP (S,R)) (Scheme 3) 1.(3S,9R)-3-[(R)-3-Dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradecanoylamino]-decan-1,10-diol(C-15)

To a solution of(2R)-5-amino-2-[(R)-3-benzyloxytetradecanoylamino]pentan-1-ol (PCTWO00146127A1) (8 g, 18.4 mmol) in anhydrous CH₂Cl₂ (60 mL) at 25° C. wasadded compound C-3 (9 g, 17.5 mmol) in anhydrous CH₂Cl₂ (28 mL). Thesolution was stirred for 3 days at 20-21° C. The suspension was dilutedwith CH₂Cl₂ (66 mL) and warmed up to 30° C. to obtain a clear solution.Acetonitrile (320 mL) was slowly added to the solution. The solution wascooled down to 20° C. and stirred for 2 H. The precipitate was filtered,washed with acetonitrile and dried under vacuum to obtain C-15 (13.7 g,83%) as a white solid. mp=103° C.

2.(3S,9R)-3-[(R)-3-Dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradecanoylamino]-decan-1,10-diol1,10-bis-(dibenzyl phosphate) (C-16)

To a solution of C-15 (1.02 g; 1.08 mmol) and 1H-tetrazole (454 mg; 6.48mmol; 6 eq) in anhydrous THF (46 mL) at room temperature and under argonwas added N,N-dibenzyl diethylphosphoramidite (85%, 1.50 mL; 4.97 mmol;4.6 eq). After 30 min under stirring, the reaction mixture was cooleddown to −20° C., then a solution of mCPBA (57-86%; 1.32 g; 7.67 mmol;7.4 eq) in CH₂CO₂ (30 mL) was added. After 45 min at −20° C., thesolution was warmed up to 0° C. and a saturated solution of Na₂S₂O₃ (25mL) was added and the mixture was stirred for 10 min. The solution wasdiluted with ether, the organic phase was separated and washed withsaturated Na₂S₂O₃ (5×), then with saturated NaHCO₃ (2×). The organicphase was dried over MgSO₄, filtered and concentrated. Purification byflash chromatography on silica gel (CH₂Cl₂/acetone 4/1 then 3/1) gaveC-16 (1.38 g; 87%) as colorless oil.

3.(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol1,10-bis-dihydrogenophosphate) (C-17) (=OM-294-DP (S,R))

A solution of compound C-16 (2.7 g, 1.84 mmol) in isopropanol (150 mL)was hydrogenated over 10% Pd on C (320 mg) at room temperature and underatmospheric pressure of hydrogen for 3 h. The catalyst was removed byfiltration through a millipore membrane. The filtrate was concentratedand dried under high vacuum to give C-17 (1.8 g; 98%) as an amorphoussolid.

Example 4 N-[(R)-3-dodecanoyloxytetradecanoylamino]-L-aspartic acid,α-N-{(4R)-5-hydroxy-4-[(R)-3-hydroxytetradecanoylamino]-pentyl}amide5-O-(6, 7-dihydroxyheptanoate) (=OM-197-MC-HD (S,R)) (Scheme 4) 1.N-[(R)-3-dodecanoyloxytetradecanoylamino]-L-aspartic acid,α-N-{(4R)-5-hydroxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}amide-β-benzylester, 5-O-(6,7-isopropylideneheptanoate) (C-18)

To a solution of the compoundN-[(R)-3-dodecanoyloxytetradecanoylamino]-L-aspartic acid,α-N-{(4R)-5-hydroxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}amide-β-benzyl ester (PCT WO00146127A1) (1.14 g, 1.09 mmol.) andcompound 6,7-Isopropylideneheptanoic acid (C-12) (487 mg, 2.48 mmol.) indry CH₂Cl₂ (30 mL) at 0° C. and under argon flow, there are added insuccession commercially available1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (485 mg,2.53 mmol.) and 4-dimethylaminopyridine (43 mg, 0.35 mmol). The reactionmixture is then stirred for 30 minutes at 0° C. and thereafter overnightat room temperature. The reaction medium is then washed with water and asolution of 1N HCl followed by layer separation. The organic layer isdried over MgSO₄, filtered and evaporated. By running a flashchromatography purification on a silica gel (CH₂Cl₂/Acetone, 9:1), thereis recovered the coupling reaction product C-18 (1.20 g; 76%) as a whitesolid.

ES/MS: m/z 1255 [M+Na]⁺.

2. N-[(R)-3-dodecanoyloxytetradecanoylamino]-L-aspartic acid,α-N-{(4R)-5-hydroxy-4-[(R)-3-hydroxytetradecanoylamino]-pentyl}amide5-O-(6,7-dihydroxyheptanoate) (C-19) (=OM-197-MC-HD (S,R))

A solution of the compound C-18 (1.20 g, 0.97 mmol.) in a 2/1CH₂Cl₂/ethanol mixture (50 mL) containing acetic acid (5 mL) ishydrogenated in presence of Pd on carbon containing 10% Pd at roomtemperature and under atmospheric pressure hydrogen for 12 to 36 hours.The catalyst is filtered off. The filtrate is evaporated to dryness andthe residue is then dried by suction from a vacuum pump to obtain C-19(1 g, quantitative yield). ES/MS: m/z 1012 [M+H]⁺; 1034 [M+Na]⁺.

Example 5(5S,11R)-1-Amino-5-[(R)-3-dodecanoyloxytetradecanoylamino]-6-oxo-7-aza-11-[(R)-3-hydroxytetradecanoylamino]-dodecan-12-ol12-dihydrogenophosphate (=OM-294-BA-MP (S,R)) (Scheme 5) 1.N^(α)—[(R)-3-Dodecanoyloxytetradecanoyl]N^(ε)-benzyloxycarbonyl-L-lysine (C-20)

To a solution of (R)-3-dodecanoyloxytetradecanoic acid P-109 [Bull.Chem. Soc. Jpn 60 (1987), 2205-2214] (153 mg; 0.36 mmol) in anhydrousTHF (4 ml) at −15° C. and under argon were added successivelyN-methylmorpholine (40 μl; 0.36 mmol 1 eq) and isobutyl chloroformate(47 μl; 0.36 mmol; 1 eq). After 30 min under stirring at −15° C., asolution of commercially available H-L-Lysine(Z)-OH (100 mg; 0.36 mmol;1 eq) in CH₃CN/H₂O (3.5/1 (4.5 mL) containing Et₃N (0.16 mL) was added.The reaction mixture was stirred for 20 h at room temperature. Theorganic solvent was then evaporated and the aqueous layer was cooleddown to 0° C., acidified with a 10% aqueous solution of citric acid downto pH=3 and extracted with AcOEt (3×). The organic layer was dried overMgSO₄, filtered and evaporated. By running a flash chromatographypurification on a silica gel (1/1 petroleum ether/AcOEt eluentcontaining 0.25% of acetic acid) followed by coevaporation of toluenegave C-20 (172 mg; 70%) as a white crystalline solid. MS: (IS+) m/z690.0 [M+H]⁺, 707.0 [M+NH₄]⁺, 712.0 [M+Na]⁺, 727.5 [M+K]⁺. ¹³C-NMR(62.89 MHz, CDCl₃), δ in ppm: 174.90, 173.73, 170.66, 156.98, 136.53,128.57, 128.17, 128.05, 71.27, 66.79, 52.24, 41.32, 40.47, 34.59, 34.24,31.99, 31.45, 29.72, 29.43, 29.25, 25.29, 25.07, 22.76, 22.27, 14.19.

2.(5S,11R)-1-Benzyloxycarbonylamino-5-[(R)-3-dodecanoyloxytetradecanoyl-amino]-6-oxo-7-aza-11-[(R)-3-benzyloxytetradecanoylamino]-dodecan-12-ol(C-21).

To a solution of C-20 (150 mg; 0.22 mmol.) and(2R)-5-amino-2-[(R)-3-benzyloxy-tetradecanoylamino]pentan-1-ol WZ-10b(PCT WO00146127 A1) (95 mg; 0.22 mmol.; 1.0 eq) in anhydrous CH₂Cl₂ (3ml) at 0° C. was added commercially available HOAt(1-hydroxy-7-azabenzotriazol) (36 mg, 0.26 mmol., 1.2 eq.) andcommercially-available N,N′-diisopropylcarbodiimide (41 μl, 0.22 mmol.,1.2 eq.). The reaction mixture was stirred for 1 hour at 0° C. andthereafter overnight at room temperature. The reaction mixture wassubsequently washed with water, a 1N HCl solution, and a saturatedsolution of NaHCO₃ followed by layer separation. The organic layer wasdried on MgSO₄, filtered and evaporated. By running a flashchromatography purification on a silica gel (3/1 CH₂Cl₂/acetone eluent),there was recovered C-21 (165 mg; 68%) as a white crystalline solid. MS:(IS+) m/z 1105.8 [M+H]⁺, 1128.0 [M+Na]⁺, ¹³C-NMR (62.89 MHz, CDCl₃), 6in ppm: 173.56, 172.20, 171.87, 170.23, 156.82, 138.32, 136.69, 128.53,128.47, 128.09, 128.00, 127.91, 127.82, 76.77, 71.48, 71.15, 66.59,64.77, 53.1, 51.39, 41.71, 41.63, 40.46, 39.45, 34.57, 34.51, 34.12,31.97, 29.70, 29.51, 29.41, 29.25, 28.69, 25.38, 25.29, 25.19, 25.09,22.74, 22.50, 14.18.

3.(5S,11R)-1-Benzyloxycarbonylamino-5-[(R)-3-dodecanoyloxytetradecanoylamino]-6-oxo-7-aza-11-[(R)-3-benzyloxytetradecanoylamino]-dodecan-12-ol12-dibenzyl phosphate (C-22)

To a solution of C-21 (150 mg; 0.14 mmol) and 1H-tetrazole (29 mg; 0.41mmol; 3, eq) in anhydrous THF (8 mL) at room temperature and under argonwas added N,N-dibenzyl diethylphosphoramidite (85%, 110 μL; 0.31 mmol;2.3 eq). After 30 min under stirring, the reaction mixture was cooleddown to −20° C., then a solution of mCPBA (57-86%; 87 g; 0.50 mmol; 3.7eq) in CH₂Cl₂ (6 mL) was added. After 45 min at −20° C., the solutionwas warmed up to 0° C. and a saturated solution of Na₂S₂O₃ (5 mL) wasadded and the mixture was stirred for 10 min. The solution was dilutedwith ether, the organic phase was separated and washed with saturatedNa₂S₂O₃ (5×), then with saturated NaHCO₃ (2×). The organic phase wasdried over MgSO₄, filtered and concentrated. Purification by flashchromatography on silica gel (CH₂Cl₂/acetone 4/1 then 2/1) gave C-22(149 mg; 81%) as a amorphous solid. MS: (IS+) m/z 1366.0 [M+H]⁺, 1383.5[M+NH₄]⁺, 1388.5 [M+Na]⁺, 1088.0 [M-(BnO)₂OPOH)+H]⁺, ¹³C-NMR (62.89 MHz,CDCl₃), δ in ppm: 173.51, 171.77, 171.27, 169.95, 156.67, 138.32,136.69, 135.66 (d), 135.55 (d), 128.69, 128.51, 128.43, 128.03, 127.77,127.69, 76.49, 71.28, 71.20, 69.66 (d), 69.58 (d), 68.72 (d), 66.51,52.82, 48.62 (d), 41.76, 41.46, 40.46, 39.01, 34.54, 34.06, 31.96,29.68, 29.49, 29.39, 29.22, 27.95, 25.29, 25.18, 25.05, 22.73, 22.45,14.17.

4.(5S,11R)-1-Amino-5-[(R)-3-dodecanoyloxytetradecanoylamino]-6-oxo-7-aza-11-[(R)-3-hydroxytetradecanoylamino]-dodecan-12-ol12-dihydrogenophosphate (C-23) (=OM-294-BA-MP (S,R))

A solution of the compound C-22 (130 mg, 0.095 mmol.) in a 3/1CH₂Cl₂/ethanol mixture (20 mL) containing acetic acid (3 mL) ishydrogenated in presence of Pd on carbon containing 10% Pd at roomtemperature and under atmospheric pressure hydrogen for 36 hours. Thecatalyst is filtered off. The filtrate is evaporated to dryness and theresidue is then dried by suction from a vacuum pump to obtain C-23 (84mg, 91%). MS: (IS+) m/z 962.0 [M+H]⁺, 984.0 [M+Na]⁺.

Biological Activities of the Compounds According to the Invention

Example 6 In vitro Regulatory Effect of OM-197-MP-AC and OM-294-DP onLPS Induced Inflammatory (IL-12 and TNF-α) and Anti-inflammatory (IL-10)Cytokines on Murine DC Cells

Protocol:

Bone marrow cells were isolated and cultured as described below:

Femora and tibiae from two male C57BL/7, 6 week-old (Charles RiverLaboratories France), were removed, stripped of muscles and tendons, anddesinfected with 70% ethanol. Both ends of each bone were cut and themarrow was flushed with HBSS (Gibco BRL) using a syringe with a 26-gaugeneedle. The resulting cell suspension was centrifuged for 5 min. at 500g and washed in HBSS. The cells were resuspended at 3×10⁵ cells/ml inRPMI-1640 (Gibco BRL) supplemented with 2 mM L-glutamin (Sigma), 100U/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma), 50 μMβ-mercaptoethanol (Sigma), 10% heat-inactivated FCS (Sigma) and 15 ng/mlmurine rGM-CSF (R&D).

To generate bone marrow (BM) derived dendritic cells (DC), BM leukocyteswere cultured in 100-mm bacteriological petri dishes (Falcon 1029) for 8days at 37° C. in 5% CO₂. On day 0, 10 ml of cell suspension were seededper dish. On day 3, another 10 ml of freshly prepared medium were addedto each plate and on day 6, 9 ml of top medium were carefully removedfrom each plate and replaced by 10 ml of fresh medium.

Non-adherent cells of the 8-day old BM cell cultures were collected,centrifuged for 5 min. at 500 g, and resuspended at 1.25×10⁶ cells/ml insupplemented RPMI containing 10 ng/ml GM-CSF. The cells were then seededin 24-well tissue culture plates (1×10⁶ cells/well) and incubated for 1h 30 at 37° C. in 5% CO₂ in the absence or presence of OM-294-DP orOM-197-MP-AC (0.01-100 μg/ml/200 μl/well). LPS (E. coli O26:B6, Sigma),was then added at 1 μg/ml, and the plates were further incubated. After20 h incubation, the supernatants were collected for analysis ofcytokines. IL-12p70, IL-10, and TNF-α concentrations in DC culturesupernatants were measured by ELISA using OptElA matched Ab pairs fromBD Pharmingen. Supersignal ELISA Pico chemiluminescent peroxidasesubstrate (BD Pharmingen) was used for the detection by luminometry(Wallac 1420 multilabel counter Victor-2). The assays were performedaccording to manufacturer's instructions.

Results:

-   a) Effect of OM-197-MP-AC and OM-294-DP on LPS-Induced IL-12 and    IL-10 Production.

The results on LPS-induced IL-12 and IL-10 production are shown in theFIG. 1.

-   b) Effect of OM-197-MP-AC and OM-294-DP on LPS-Induced TNF-α    Production.

The results on LPS-induced TNF-α production are shown in the FIG. 2.

Conclusion:

The above results clearly show that both OM-197-MP-AC and OM-294-DPdecrease the LPS-induced production of the inflammatory cytokines (IL-12and TNF-α), and in striking contrast increase the production of theanti-inflammatory cytokine IL-10, suggesting that the compounds of theinvention could act as therapeutic anti-inflammatory agents.

Example 7 Direct Effects of a Series of Four Triacylated Derivatives onHuman CD4⁺ T Cells Protocol

A series of four compounds of the present invention (OM-197-MP-AC,OM-197-MC-HD, OM-294-BA-MP, OM-197-MP-HD) were tested directly assummarized below: Naïve CD4⁺ T cells were magnetically sorted fromhealthy blood donor (buffy coat). The cells were stimulated in thepresence or not of graded doses of triacyl derivatives (0.02; 0.2; 2 and20 μg/ml) using plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1μg/ml). After 6 days, cytokine levels (IFN-γ and IL-13) were tested inthe culture supernatants using ELISA.

Results:

The results for the cytokines production are shown in the FIG. 3. T cellproliferation was also tested but was not modified be the compounds ofthe invention.

Conclusion:

IL-13 production (white histograms) by CD4⁺ T cells is reduced in thepresence of OM-197-MP-AC, OM-197-MC-HD, OM-294-BA-MP, OM-197-MP-HDwhereas IFN-γ production (dark histograms) and T cell proliferationremain unaffected. This represents a shift towards a Th1 response in thepresence of the four triacylated molecules tested.

Example 8 Effect of OM-197-MP-AC in a Model of Asthma

Since in the previous example it has been shown that the triacylatedmolecules tested decreased the production of a cytokine likely involvedin the pathology of asthma (i.e. IL-13) the inventors aimed here atinvestigating in vivo the effects of a member of the series(OM-197-MP-AC) in the modulation of Th2 differentiation of naïve Thelper cells and the subsequent development of allergic asthma.

In the present study, the inventors have investigated first (part a) theeffect of OM-197-MP-AC given during the sensitization phase on thedevelopment of LACK-induced allergic asthma, and then the effect of themolecule tested therapeutically (part b).

Part a) “Prophylactic” Treatment of Asthma

Protocol:

Mice and Induction of Allergic Asthma:

Females Balb/c ByJ mice of 6-week old were purchased from the Centred'Elevage Janvier (CERJ, Le Genest-St-lsle, France). All the mice weresensitised by 2 i.p. injections of 10 μg LACK protein precipitated in 2mg of Alum (PerBio Science France SAS, Brebières, France) at days 0 and7.

Animals were divided in groups, as follow:

-   -   LACK-sensitized and saline-challenged mice (4 mice)    -   LACK-sensitized and challenged mice (8 mice)    -   OM-197-MP-AC-treated, LACK-sensitized and challenged mice (8        mice)

Mice of the third group were treated intraperitoneally with 1 mg/Kg (20μg per mouse) of OM-197-M P-AC. Mice were treated on days: −1, 1, 2, 3,6, 8, 9, 10 and 11. From day 16 to day 21, mice of groups B and C wereexposed to a daily 20-min aerosol challenge of a LACK solution (0.15%)whereas mice of the saline group received a saline solution as control.

Airway Hyperresponsiveness (AHR):

AHR was measured for all the mice one day after the last antigenchallenge by whole-body plethysmography (Emka) in response to increasingconcentrations (6-25 mg/ml) of inhaled methacholine (acetyl methylcholine, Sigma). AHR is expressed as enhanced pause (Penh, see FIG. 4),a dimensionless parameter perfectly correlated to airway compliance andresistance calculated value, which correlates with measurement of airwayresistance, impedance, and intrapleural pressure.

Reagents:

-   -   LACK recombinant protein was produced in E. coli and purified as        described by Mougneau et al., 1995.    -   OM-197-MP-AC, batch DL040906 at the concentration of 0.98 mg/ml,        was from OM PHARMA.    -   Anti-IgE EM-95 mAbs were a gift from DNAX (Palo Alto, Calif.,        USA).    -   Methacholine (acetyl methyl choline) was purchased from Sigma        (Saint Quentin Fallavier, France).    -   CBA array beads (Flex set) were purchased from BD Biosciences.        Antibody Titers:

All the mice were bled by heart puncture one day after the last aerosol.Total IgE were quantified by ELISA using rat EM-95 anti-IgE mAbs ascoating antibody and rat anti-IgE mAbs coupled to biotin as secondantibody as described (Julia et al., 2002).

Analysis of Bronchoalveolar Lavage (BAL) Cells (Percentage ofEosinophils):

Individual mice were bled and a canula was inserted into their trachea.Lungs were washed 3 times with 1 ml of warmed PBS. Cells were washedwith PBS, resuspended in 300 μl, and counted using a Burker-Türkchamber. For differential BAL cell counts, cytospin preparations weremade and stained with Wright/Giemsa coloration. The respective numbersof neutrophils, eosinophils, and other mononuclear cells were determinedby microscopic examination. Only the percentage of eosinophils (FIG. 5)is reported here.

Analysis of Lung Cytokine Contents:

To investigate lung cytokine contents of treated and untreated animals,protein extracts were prepared from lungs of LACK-sensitized andPBS-challenged wt mice (group “Control”), LACK-sensitized and challengedwt mice (group Asthma”), OM-197-MP-AC-treated wt mice (group “OM-197”).IL-4, IL-5, IL-13 contents were analyzed by multiplex analysis usingFACSArray.

Results:

Measurement of Airway Hyperresponsiveness (AHR):

Treated or untreated LACK-sensitized mice (see above) were nextchallenged with daily aerosol of LACK for five consecutive days. One dayafter the last aerosol, AHR was measured by whole-body plethysmographyin response to increasing concentrations of aerosolized methacholine.

As expected, LACK-sensitized and saline-challenged mice did not developAHR when exposed to methacholine whereas untreated LACK-sensitized andchallenged mice developed a strong AHR, as reflected by high Penh values(FIG. 4). In sharp contrast, LACK-sensitized and challenged mice thatwere treated with OM-197-MP-AC did not exhibit AHR.

Conclusion:

Airway hyper-responsiveness on exposure of mice to aerosol antigen,measured in the presence of methacholine, was reduced back to controllevels (saline) by treatment with OM-197-MP-AC.

Characterization of the Percentage of Eosinophils in Broncho-AlveolarRavages (BAL):

The results are reported in FIG. 5.

LACK-sensitized mice that received saline aerosols, did not exhibiteosinophils in BAL whereas LACK-sensitized mice that received LACKaerosol challenges did. In addition, OM-197-MP-AC-treated mice exhibited3-times less eosinophils, than untreated control mice (FIG. 5).

Conclusion:

Treatments with OM-197-MP-AC strongly decreased BAL eosinophilia.

Measurement of Immunoglobulins (Ig) in Sera:

The results for IgE are shown in FIG. 6:

Titers of total IgE (FIG. 6) were significantly decreased when mice weretreated with OM-197-MP-AC compound.

Conclusion:

Thus, sera of mice treated with OM-197-MP-AC contained 2-fold less totalIgE as compared to sera of untreated sensitized, challenged mice (i.e.“asthmatic animals”).

Measurement of Lung Cytokines:

To further investigate the effects of prophylactic OM-197-MP-AC onallergic airway inflammation, proteins were extracted from lungs of bothtreated and untreated WT mice, and cytokines known to play a importantrole in asthma (IL-4, IL-5 and IL-13) were quantified by multiplexanalysis using CBA array beads and FACSarray. Data were normalized tothe total quantity of proteins and given as pg of cytokine per mg oflung proteins.

While the amounts of IL-4, IL-13, and IL-5 were under the limit ofdetection in lungs of LACK-sensitized and PBS-challenged mice (see“Control” in FIG. 7) the amounts of these cytokines increaseddramatically upon LACK challenges (see “Asthma” in FIG. 7). In contrast,lungs of LACK-sensitized and challenged mice that have been treated withOM-197-MP-AC contained 5 times less IL-4 (p=0.0003) 5 times less IL-5(p=0.0003); and 3.5 times less IL-13 (p=0.003) than untreated mice (see“OM-197” in FIG. 7).

Beside IL-4, IL-5, and IL-13, other cytokines were quantified bymultiplex analysis using CBA array beads and FACSarray. The main resultsare reported herebelow. IFN-γ was also found to be upregulated in lungsof LACK-sensitized and challenged mice as compared to LACK-sensitizedand PBS-challenged mice. Lungs of OM-197-MP-AC-treated mice containedboth 2-times less IFN-γ than those of untreated mice. KC is thefunctional homologous chemokine of human IL-8 or CXCL8, and binds toCXCR2 expressed by both neutrophils and eosinophils. Indeed, KC mayallow the recruitment of granulocytes to inflamed tissues. While KC waspresent in very low quantity in lungs of LACK-sensitized andPBS-challenged mice, its expression highly increased upon LACK aerosolchallenges. The amount of KC produced in lungs was reduced by half upontreatment with OM-197-MP-AC.

The pro-inflammatory cytokine, IL-6 was found in lungs ofLACK-sensitized and PBS-challenged and slightly increased upon LACKaerosols. Lungs of OM-197-MP-AC-treated mice exhibited 1.5-times lessIL-6 than those of untreated mice. The chemokine MCP-1 was found to beexpressed in lungs of sensitized and PBS-challenged mice and wasslightly upregulated upon challenge. Lungs of mice that were treatedwith OM-197-MP-AC contained 1.8 times less MCP-1 than those of untreatedanimals.

The amounts of TNF-α in lungs of treated and untreated mice did notyield significant differences.

IL-9 was not detected in any of the samples.

Taken together these data clearly indicate that prophylacticOM-197-MP-AC induces not only a strong decrease of Th2 cytokines thatare crucial for inducing allergic airway inflammation, and AHR, but alsoa decrease of other cytokines and chemokine which contribute to the lunginflammation and to the recruitment of inflammatory effector cells suchas eosinophils.

General Conclusion for Example 8 a:

This study clearly demonstrated a protective role of one the moleculesof the invention, OM-197-MP-AC, in the development of allergic asthma.Treatments with OM-197-MP-AC starting just before the sensitizationphase inhibited the development of AHR, and strongly decreased BALeosinophilia. In addition, the amounts of total IgE were decreased inthe serum of mice that had been treated with OM-197-MP-AC.

The data also show that OM-197-MP-AC treatments impaired mainly thedevelopment of cytokines involved in asthma, and the secretion of otherinflammatory cytokines. One possible mechanism would be thatOM-197-MP-AC might induce a specific immunosuppressive response thatwould control Th2 development, and subsequent airway inflammation.

Part B) “Therapeutic” Treatment of Asthma

Protocol:

The Main Differences Compared to the Previous (Part a) Protocol are:

-   -   Mice were treated with OM-197-MP-AC i.p. only on days 15, 17 and        19 (i.e. at least 1 week after the second sensitization) at the        dose of 1 mg/Kg (20 μg per mouse).    -   The lung cytokines measured by multiplex analysis using        FACSArray were IL-4, IL-5, IL-13, IFN-γ, and IL-10.        Results:        Total Cell number and eosinophils in BAL:

Two days after the last aerosol, mice were sacrificed and BAL cellsharvested. As expected, untreated LACK-sensitized mice exhibited amassive influx of cells in BAL upon LACK challenges (20-fold more cellsas compared to PBS-challenged mice) (FIG. 8). Very interestingly, BALcell recruitment was impaired by 90% upon therapeutic administrationswith OM-197-MP-AC (p<0.01) (FIG. 9), with 40-45-fold less eosinophils ascompared to untreated mice.

Conclusion:

OM-197-MP-AC provided three times therapeutically was able todramatically decrease pulmonar cellular extravasion and eosinophilia inBAL.

Measurements of Lung Cytokines.

Since airway eosinophilia was so drastically reduced upon therapeutictreatment with OM-197-MP-AC, we sought of analyzing IL-4, IL-5, IL-13,IL-10 and IFN-γ, lung contents (FIG. 10). Indeed, when compared to lungsof untreated mice, the amounts of the Th2-cytokines: IL-4, IL-5 andIL-13 were strongly reduced in lungs of treated mice. IL-4 levels werereduced by 90%, IL-13 amounts by 85%, and IL-5 amounts by 70% upontreatment with OM-197-MP-AC (from p<0.00005 to 0.005).

Conclusion:

Clearly OM-197-MP-AC when provided therapeutically only three times wasable to decrease the level of Th2 cytokines. This was not due to a shifttowards Th1 cytokine since IFN-γ levels remained low (1.5 to 3 pg/ml)for all mice and were reduced in treated mice. The amounts of IL-10 thatis both a Th2 and an immunosuppressive cytokine, were low, and were notsignificantly increased upon treatment

Measurement of IgE Levels:

In order to further characterize the immune status of mice aftertherapeutic treatment with OM-197-MP-AC, we analyzed LACK-specific IgEin sera of treated mice and in those of untreated mice by ELISA. WhereasLACK-specific IgE levels increased 7-fold upon exposition to LACKaerosols, sera of OM-197-MP-AC-treated mice contained 2-fold less(p<0.05) LACK-specific IgE compared to untreated LACK challenged mice.

Conclusion:

Clearly OM-197-MP-AC when provided therapeutically decreased sericLACK-specific IgE levels.

General Conclusion for Example 8b.

We demonstrated here, that the product of the invention, OM-197-MP-AC,when provided therapeutically, clearly decreased eosinophilia, as wellas the level of Th2 cytokines such as IL-4, IL-5, and IL-13, and alsothe level of the allergen-specific IgE

General Conclusion for Example 8

Clearly OM-197-MP-AC was active both preventively and therapeutically invivo in a model of asthma, as judged by its effects on manyasthma-relevant read-outs.

Example 9 Effect of OM-197-MP-AC by Other Routes of Administration inthe Murine Model of Asthma

The results presented in example 8 show that OM-197-MP-AC is effectivein the prevention of allergic responses in the murine LACK model ofasthma, when administered by the i.p. route. Other ways ofadministration, more compatible with the human usage, were thereforeinvestigated in this model (intranasal, intragastric, subcutaneous).

Protocol:

43 females BALB/c ByJ mice 6-week old were purchased from the Centred'Elevage Janvier (CERJ, Le Genest-St-lsle, France). All the mice weresensitised by 2 i.p. injections of 10 μg LACK protein precipitated in 2mg of Alum (PerBio Science France SAS, Brebières, France) at days 1 and8 (Julia et al., 2002). OM-197-MP-AC was given from day 0 to day 12 asdescribed below. All the mice were challenged from day 16 to day 20 witheither a solution of LACK (0.15%) or with PBS as indicated below.

Experimental groups were the following:

-   -   Group A: untreated, LACK-sensitised and PBS-challenged mice (3        mice)    -   Group B: untreated LACK-sensitised and -challenged mice (7 mice)    -   Group C: i.p. OM-197-MP-AC-treated, LACK-sensitised and        -challenged mice (5 mice)    -   Group D: i.g. OM-197-MP-AC-treated, LACK-sensitised and        -challenged mice (7 mice)    -   Group E: s.c. OM-197-MP-AC-treated, LACK-sensitised and        -challenged mice (7 mice).    -   Group F: i.n. OM-197-MP-AC-treated, LACK-sensitised and        -challenged mice (7 mice)    -   Group G: aerosols OM-197-MP-AC-treated, LACK-sensitised and        -challenged mice (7 mice)

Mice of groups C, D, E, and G were treated on days: 0, 2, 3, 4, 7, 9,10, 11, whereas mice of group F (intranasal route) were only treated ondays 0, 4, 7, and 11.

Mice of groups C and E were treated i.p. and s.c. respectively with 1mg/Kg (20 μg per mouse) of OM-197-MP-AC.

Mice of group D were treated intragastrically with 50 mg/Kg (1 mg permouse) of OM-197-MP-AC.

Mice of group F were treated i.n. with 20 μg of OM-197-MP-AC in a volumeof 20 μl (OM-197-MP-AC at 2 mg/ml, 10 μl/nostril.)

Mice of group G received aerosols of a solution of OM-197-MP-AC at 0.2%(2 mg/ml) for 10 minutes.

Reagents:

-   -   LACK recombinant protein was produced in E. coli and purified as        described (Mougneau et al., 1995).    -   OM-197-MP-AC, batch #050323, at the concentration of 2.2 mg/ml,        and batch #050322, at the concentration of 2.17 mg/ml were        provided by OM PHARMA, and used for oral and all the other        administrations, respectively.    -   Anti-IgE (R35-118) coupled to biotin, was purchased from BD        Biosciences (Le Pont de Claix, France). Anti-IgE EM-95 mAbs were        a gift from DNAX (Palo Alto, Calif., USA).    -   Intranasal treatment was performed after light anaesthesia of        the mice using Isoflurane (Aerrane, Baxter).        Antibody Titers:

All the mice were bled by heart puncture 2 days after the last aerosol.Total IgE were quantified by ELISA using rat EM-95 anti-IgE mAbs ascoating antibody and rat anti-IgE mAbs coupled to biotin as secondantibody as described elsewhere (Julia et al., 2002).

Analysis of Bronchoalveolar Lavage (BAL) Cells:

Individual mice were bled and a canula was inserted into their trachea.Lungs were washed 3 times with 1 ml of warmed PBS. Cells were washedwith PBS, resuspended in 300 μl, and counted using a Burker-Türkchamber. For differential BAL cell counts, cytospin preparations weremade and stained with Wright/Giemsa.

Results:

-   a) The respective numbers of neutrophils, eosinophils, lymphocytes,    and other mononuclear cells were determined by microscopic    examination and are reported in FIG. 11 for each route of    administration tested.

As the inventors had previously reported in example 8, mice treated i.p.with OM-197-MP-AC exhibited both reduced percentage and reduced numbersof eosinophils in BAL. However, BAL cell contents of mice treated s.c.and i.g with OM-197-MP-AC did not yield differences with those ofuntreated mice. In contrast, both eosinophil frequencies (not shown) andnumbers (FIG. 11) were reduced by half in BAL of mice that had receivedi.n. OM-197-MP-AC. In addition, mice treated with OM-197-MP-AC aerosolsexhibited the same tendency as i.n. treated animals.

Conclusion:

At least OM-197-MP-AC was significantly efficient in both the i.p. andthe intranasal routes to significantly diminish in this model the numberof eosinophils in the bronchoalveolar lavages of mice sensitized to anallergen.

-   b) Then, plasmatic IgE levels were measured as described in the    method section. In agreement to our previous data (example 8), we    found that mice that had been treated i.p. with OM-197-MP-AC    exhibited reduced amounts of total IgE (FIG. 12). In addition, mice    that had received i.n. injections of OM-197-MP-AC also exhibited    reduced titers of total serum IgE. In contrast, mice treated i.g.,    s.c. or with aerosols presented similar amounts of total serum IgE    as compared to untreated mice.    Conclusion:

OM-197-MP-AC administered either by the i.p. or by the intranasal routedecreased significantly total IgE levels in this model of asthma.

Example 10 Increased Effect of Formulated OM-197-MP-AC by the Oral Routeof Administration in the Murine Model of Asthma

The results presented in example 9 show that OM-197-MP-AC is ineffectivein the prevention of allergic responses in the murine LACK model ofasthma, when administered unformulated by the oral route. An assay offormulation in order to increase the activity of OM-197-MP-AC by theoral route was therefore investigated in this model. Therefore in thisstudy, we investigated whether the triacylated OM PHARMA compoundOM-197-MP-AC would protect mice against allergic airway inflammationwhen provided as a gastro-intestinal resistant formulation (OM-197-MP-ACwith Lutrol® F 127, also known as poloxamer 407).

Protocol:

The OM-197-MP-AC/Lutrol® F 127 formulation was prepared by mixing 12.875ml of a solution of OM-197-MP-AC at 4.35 mg/ml and 5.6 mL of Lutrol F127at 50 mg/ml. The administered volume was 330 μl.

Reagents and Equipment

-   -   Saline solution were given as control aerosols    -   Recombinant LACK protein was produced in E. coli, and purified        onto a Ni-NTA affinity column    -   Aluminium hydroxide (Alum) was purchased from Pierce    -   The cytocentrifuge used was a Cytospin 4 (Thermo-Shandon,        Cheschire, U.K.), cytoslides were purchased from Thermo-Shandon        and Wright and Giemsa stains from Sigma.    -   Aerosols were given using an ultra-son nebulizer Ultramed        (Medicalia, Forenze, Italy)    -   CBA beads array IL-4 were purchased from BD Biosciences.    -   The flow cytometer FACSarray has been purchased from BD        Biosciences.        Animals:    -   6 weeks old female BALB/c ByJ mice were purchased from The        Centre d'Elevage Janvier, France, and were kept under        specific-pathogen free conditions in the animal facility. They        were fed with a standard diet provided by Safe (Augy, France).        Protocol:

15 BAL/c mice (including the mice for the control groups A and B, shownalso in the examples 8 and 9) were used for this experiment.

-   A: untreated LACK-sensitized and saline-challenged mice (3 mice)-   B: untreated LACK-sensitized and challenged mice (6 mice)    Prophylactic Treatment-   F: OM-197-MP-AC (oral)-treated LACK-sensitized and challenged mice    (6 mice)

Mice of group F were treated orally on days 0, 2, 3, 4, 7, 9, 10 11, and12 with 1 mg of formulated OM 197 MP AC.

On day 1 and day 8, mice were sensitized i.p. with LACK/Alum. From day16 to day 20, all the groups except group A mice were challenged withaerosols of a solution of LACK (0.15%). Group A received a salinesolution (NaCl 0.9%) for 40 minutes instead.

At day 22, all the mice will be sacrificed. For all the animals, BAL,and lungs without peri-bronchial lymph nodes were harvested.

BAL cells will be counted and cell contents will be analyzed aftermicroscopic examination of cytospins following wright/giemsa staining.In addition, lungs of each group were harvested and frozen in liquidnitrogen for further cytokine content analysis. To investigate cytokinecontent, protein extracts will be prepared from lungs of LACK-sensitizedand PBS-challenged mice (group A), LACK-sensitized and challenged mice(group B), and formulated OM-197-MP-AC-treated wt mice (group F). IL-4amount was analyzed by multiplex analysis using FACSArray.

Results:

The respective numbers of neutrophils, eosinophils, lymphocytes, andother mononuclear cells were determined by microscopic examination andare reported in FIG. 13.

In contrast to the results reported in example 8 (and FIG. 11) were BALcell contents of mice that have been treated i.g with OM-197-MP-AC(unformulated), when OM-197-MP-AC was formulated with Lutrol, the numberof neutrophils, eosinophils (p<0.05), lymphocytes, and other mononuclearcells were reduced by half in BAL of mice that had received formulatedOM-197-MP-AC (see FIG. 13).

Moreover, IL-4 amounts were analyzed in lungs of treated and untreatedmice. Whereas IL-4 lung contents were very low to undetectable inPBS-challenged animals, IL-4 amounts increased 20-fold inLACK-challenged untreated control mice. Upon oral treatment withformulated OM-197-MP-AC (see FIG. 14), the amounts of IL-4 in lungsdecreased by 75% (p<0.01)

Conclusion:

When adequately formulated, oral OM-197-MP-AC was able to significantlydiminish in this model the number of eosinophils in the bronchoalveolarravages of mice sensitized to an allergen. This decrease was correlatedwith a decrease in IL-4.

Example 11 Effect of OM-197-MP-AC on Non Obese Diabetic (NOD) Mice

So far, we provided examples of the in vivo efficacy of OM-197-MP-AConly in a murine model of asthma. In this example, we will provide someevidence that OM-197-MP-AC is also active in another inflammatorypathology: diabetes.

Protocol:

We have previously shown that OM-197-MP-AC and other triacylatedmolecules decrease airway inflammation in asthmatic animals.

In this study, we investigated whether OM-197-MP-AC would be able toinduce protection, in another model of murine inflammatory disease: theNOD diabetes model (Non Obese Diabetic).

Group of 10 female 6-week old NOD mice were treated during 10 weeks witheither 0.01, 0.1, and 1 mg/kg of OM-197-MP-AC. A group of 6 female MODmice receiving 200 μl PBS i.p. 3 times a week was used as control.

In this series of experiments, the treatment was continued until themice have reached 16 weeks of age. This is the point in time whencontrol animals start developing overt diabetes.

Results in terms of diabetes incidence were compared to those obtainedin control animals. Diabetes was checked once a week by glucosuriatesting, using test strips (Glukotest®, Roche, France), twice a weekwhen diabetes appeared. Diabetes was confirmed by evaluating glycemia(>3 mg/ml) with test strips (Glucotrend®).

The occurrence of diabetes in the different experimental groups isplotted (see FIG. 15) using the Kaplan-Meier method, i.e. non parametriccumulative survival plot. The statistical comparison between the curvesis performed using the logrank (Mantel-Cox) test which provided thecorresponding χ2 values. OM-197-MP-AC at the dose of 1 mg/kg wassignificantly active (p=0.0321), and at the dose of 0.1 mg/kg thecompound of the invention exhibited a positive trend (p=0.0543).

In another experiment using a dose of OM-197-MP-AC of 0.3 mg/kg, theresults obtained at week 27 show a significant p value of 0.0362. Atweek 27, 82% of the animals were found diabetic in the control group andonly 27% in the OM-197-MP-AC treated group (0.3 mg/kg) exhibited thedisease.

Example 13 Toxicology: LAL Endotoxicity

Endotoxicity was determined first for OM-197-MP-AC and for OM-294-DP bythe Limulus Amoebocyte Lysate chromogenic test (Chromogenic—LAL ofBio-Whittaker, kit n°50-650U).

Protocol:

This test is based on activation by lipopolysaccharide (LPS) or productsof comparable structure, by an enzymatic cascade present in the LAL.This enzymatic activation is demonstrated by the splitting of achromogen linked to a peptide by a protease. The enzymatic reaction iscarried out at 37° C. and the formation of the chromogen over time ismeasured at 405 nm. The time necessary to reach 0.2 units of D.O. isrecorded and the endotoxic activity calculated in relation to a LPSstandard (standard curve). The results are expressed in EU (EndotoxinUnit) in relation to a standardized preparation of E. coli LPS (1 EUcorresponds to 0.08 ng equivalent LPS).

Results:

Both OM-197-MP-AC and OM-294-DP showed very little or no Limulusactivity in the chromogenic LAL assay.

Conclusion:

OM-197-MP-AC and OM-294-DP are very weakly endotoxic (at least 10⁶ folddecrease) when compared to LPS.

Example 14 Toxicology: Pyrogenicity in the Rabbit

Finally, OM-197-MP-AC and OM-294-DP were tested for their potentialpyrogenicity in the rabbit.

Protocol:

3 New Zealand white rabbits/group were injected i.v. with eitherOM-197-MP-AC or OM-294-DP at different doses (according to the EuropeanPharmacopoeia 2001, Method 2.6.8).

Products: OM-197-MP-AC at 0.0009, 0.009, 0.09, 0.9 mg/kg, and OM-294-DPat 0.001, 0.01, 0.1, and 1 mg/kg.

Animals: Readout: temperature increase

Results:

Both compounds were considered not pyrogenic in vivo, since thepyrogenic threshold of OM-197-MP-AC was not reached at the highest dosetested (0.9 mg/kg), and the pyrogenicity of OM-294-DP was only reachedbetween 0.01 and 0.1 mg/kg.

REFERENCES

-   Byl, B., Libin, M., Bauer, J., Martin, O. R., De Wit, D., Davies,    G., Goldman, M., and Willems, F. (2003). OM197-MP-AC induces the    maturation of human dendritic cells and promotes a primary T cell    response. Int Immunopharmacol 3, 417-425.-   Julia, V., Hessel, E. M., Malherbe, L., Glaichenhaus, N., O'Garra,    A., and Coffman, R. L. (2002). A restricted subset of dendritic    cells captures airborne antigens and remains able to activate    specific T cells long after antigen exposure. Immunity 16, 271-283.-   Mougneau, E., Altare, F., Wakil, A. E., Zheng, S., Copolla, T.,    Wang, Z.-E., Waldmann, R., Locksley, R., and Glaichenhaus, N.    (1995). Expression cloning of a Leishmania major protective T cell    antigen. Science 268, 563-566.-   Veran, J., Mohty, M., Gaugler, B., Chiavaroli, C., and Olive, D.    (2004). OM-197-MP-AC adjuvant properties: the in vitro maturation of    normal and leukemic dendritic cells in a serum-free culture model.    Immunobiology 209, 67-77.

FIGURE LEGENDS

FIG. 1: IL-12 (left panels) and IL-10 (right panels) secretion by murineDC stimulated by LPS (1 μg/ml) alone, or first during 90 minutes withthe indicated concentrations (in μg/ml) of OM-197-MP-AC (A) or 294-DP(B), and then by LPS (1 μg/ml) for 20 additional hours. The supernatantswere collected from the DC cultures and analyzed by ELISA for thepresence of IL-12 and IL-10.

FIG. 2: TNF-α secretion by murine DC stimulated by LPS (1 μg/ml) alone,or first during 90 minutes with the indicated concentrations (in μg/ml)of 294-DP (294), or of OM-197-MP-AC (197), and then by LPS (1 μg/ml) for20 additional hours. The supernatants were collected from the DCcultures and analyzed by ELISA for the presence of TNF-α.

FIG. 3: Effect of increasing doses of the four compounds tested(OM-197-MP-AC, n=5; OM-197-MC-HD, n=5; OM-294-BA-MP, n=7; OM-197-MP-HD,n=3) on IFN-γ and IL-13 production of human CD4⁺ T cells following apolyclonal activation. Results are expressed as the median (±p25/p75) ofn independent experiments and show the percentage (%) proliferation orcytokine production of treated cells versus untreated cells (consideredas 100%, see dotted line). The statistical analysis was performed usinga nonparametric unpaired Mann-Whitney t test (2-tailed).

FIG. 4: AHR by whole-body plethysmography (Emka) in response toincreasing concentrations (6-25 mg/ml) of inhaled methacholine one dayafter the last antigen challenge. The animals were treated as describedin the protocol section. Results (mean “enhanced pause value”+/−SEM) areshown for saline-challenged animals (as the negative control group,n=4), untreated LACK-challenged animals (as the positive control group,n=8), and OM-197-MP-AC-treated LACK challenged mice (n=8).

FIG. 5: Percentage of eosinophils in bronchoalveolar lavages analyzed bymicroscopic examination of cytospin preparations stained withWright/Giemsa coloration. The groups studied are the same as those usedin FIG. 4. The dotted line indicates throughout the graph the valueobtained in untreated asthmatic animals.

FIG. 6: All the mice were treated as described in the protocol section,and were bled by heart puncture one day after the last aerosol. TotalIgE were quantified by ELISA using rat EM-95 anti-IgE mAbs as coatingantibody and rat anti-IgE mAbs coupled to biotin as second antibody asdescribed (Julia et al., 2002). Each point corresponds to a singleanimal. The groups studied are the same as those used in FIGS. 4 and 5.

FIG. 7: Protein extracts (400 μl) were prepared from left lungs ofLACK-sensitized and PBS-challenged wt mice (Control), LACK-sensitizedand challenged wt mice (Asthma), OM-197-MP-AC-treated wt mice (OM-197).IL-4, IL-5 and IL-13 contents were analyzed by multiplex analysis usingFACSArray. Results are given in pg/ml. The dotted lines indicatethroughout the graphs the values obtained in untreated asthmaticanimals.

FIG. 8: Mice were therapeutically treated three times as described inthe method section, lavages were performed in individual mice bled witha canula inserted into their trachea. Lungs were washed 3 times with 1ml of warmed PBS. Cells were washed with PBS, resuspended in 300 μl, andcounted using a Burker-Türk chamber. For differential BAL cell counts,cytospin preparations were made and stained with Wright/Giemsacoloration. The groups tested were: LACK-sensitized and PBS-challengedwt mice (Control), LACK-sensitized and challenged wt mice (Asthma), andOM-197-MP-AC-treated wt mice (OM-197). The total cell number in BAL wasdetermined by microscopic examination of cytospin preparations stainedwith Wright/Giemsa coloration. The dotted line indicates throughout thegraph the value obtained in untreated asthmatic animals.

FIG. 9: Percentage of eosinophils in BAL analyzed by microscopicexamination of cytospin preparations stained with Wright/Giemsacoloration. Mice were therapeutically treated three times as describedin the method section, cells from BAL were harvested as explained inFIG. 8. The (therapeutic) groups studied are the same as those used inFIG. 8. The dotted line indicates throughout the graph the valueobtained in untreated asthmatic animals.

FIG. 10: To analyze lung cytokine contents, lungs were harvested andleft lungs were used to prepare protein extracts. 400 μl were recoveredfor each left lung. Cytokines were measured by multiplex analysis usingFACSArray, and results are given in pg/ml. The (therapeutic) groupsstudied are the same as those used in FIGS. 8 and 9. The dotted linesindicate throughout the graphs the values obtained in untreatedasthmatic animals.

FIG. 11: Number of eosinophils, neutrophils, and lymphocytes in murineBAL. Cells were obtained and washed as described in the protocolsection. For differential BAL cell counts, cytospin preparations weremade and stained with Wright/Giemsa. At least 400 cells were scored foreach slide, and the numbers of eosinophils, neutrophils, and lymphocyteswere determined by microscopic examination. The dotted line indicatesthroughout the graph the value obtained in untreated asthmatic animals.

FIG. 12: Measurement of Allergen-specific IgE in murine sera were bledby heart puncture two days after the last aerosol, and sera wereprepared. LACK-specific IgE were measured by ELISA. Results (ng/ml) fromindividual mice are reported, the mean value in each group isrepresented by a bar. *=P<0.05. The dotted line indicates throughout thegraph the value obtained in untreated asthmatic animals.

FIG. 13: Number of eosinophils, neutrophils, and lymphocytes in murineBAL. Cells were obtained and washed as described in the protocolsection. For differential BAL cell counts, cytospin preparations weremade and stained with Wright/Giemsa. At least 400 cells were scored foreach slide, and the numbers of eosinophils, neutrophils, lymphocytes,and other cells (macrophages, dendritic cells, and pnuemocytes) weredetermined by microscopic examination. The 3 groups tested weredescribed in the protocol section of example 10. The dotted lineindicates throughout the graph the value obtained in untreated asthmaticanimals.

FIG. 14: To analyze lung cytokine contents, lungs were harvested andleft lungs were used to prepare protein extracts. 400 μl were recoveredfor each left lung. IL-4 was measured by FACSArray, and results aregiven in pg/ml The 3 groups tested were described in the protocolsection of example 10. The dotted line indicates throughout the graphthe value obtained in untreated asthmatic animals.

FIG. 15: Occurrence of diabetes in NOD mice. Mice were treated i.p. witheither PBS (6 animals), or the 3 indicated doses of OM-197-MP-AC (3groups of 10 animals). Diabetes was checked once a week by glucosuriatesting (Glukotest), and twice a week when diabetes appeared. Diabeteswas confirmed by evaluating glycemia (>3 mg/ml) with test strips(Glucotrend®). The occurrence of diabetes in the different experimentalgroups is plotted (see FIG. 15) using the Kaplan-Meier method, i.e. nonparametric cumulative survival plot. The statistical comparison betweenthe curves is performed using the logrank (Mantel-Cox) test whichprovided the corresponding χ2 values: OM-197-MP-AC 1 mg/kg p=0.321; 0.1mg/kg 0.0543.

1. A method for treating warm-blooded animals, including humans,suffering from asthma or allergic rhinitis, comprising theadministration to a patient in need thereof of an appropriate amount ofa pharmaceutical composition comprising at least one immunomodulatorycompound of the following general formula (I):

Wherein: m and n, independently from each other, are an integer rangingfrom 1 to 4, X and Y each designate a group either in neutral or chargedstate, selected from the following groups: carboxyl —COOH,dihydroxyphosphoryloxy —O—P(O) (OH)₂, hydroxysulfonyloxy —O—SO₂(OH),amino —NH₂, hydroxyl —OH, [amino(C₁-C₁₀)alkyl]aminocarbonyl—CONH(CH₂)_(n1)—NH₂, with n₁ being an integer from 1 to 10,[dicarboxy(C₁-C₅)alkyl]aminocarbonyl —CO—NH—CH(COOH)—(CH₂)_(n1)—COOH,with n₁ being an integer from 1 to 5,{carboxy[amino(C₁-C₅)alkyl]}aminocarbonyl—CO—NH—CH(COOH)—(CH₂)_(n1)—NH₂, with n₁ being an integer from 1 to 5,amino(C₁-C₁₅)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being an integerfrom 1 to 15, dihydroxy (C₁-C₁₀) alkanoyloxy—O—CO—(CH₂)_(n1)—CHOH—CH₂OH, with n₁ being an integer from 1 to 10,hydroxy(C₁-C₁₀)alkanoyloxy —O—CO—(CH₂)_(n1)—OH, with n₁ being an integerfrom 1 to 10, carboxy(C₁-C₁₀)alkanoyloxy —O—CO—(CH₂)_(n1)—COOH, with n₁being an integer from 1 to 10, oxo(C₁-C₅)alkanoyloxy—O—CO—(CH₂)_(n1)—CHO, with n₁ being an integer from 1 to 5,[carboxy(C₁-C₁₀)alkanoyl]amino(C₁-C₁₅)alkanoyloxy—O—CO—(CH₂)_(n1)—NH—CO—(CH₂)_(n2)-COOH, with n₁ being an integer from 1to 10, and n₂ being an integer from 1 to 15, R₁ and R₂ each designate anacyl group derived from a saturated or unsaturated, straight-orbranched-chain carboxylic acid having from 2 to 18 carbon atoms, whichis unsubstituted or bears one to three substituents selected amonghydroxyl, dihydroxyphosphoryloxy, alkyl of 2 to 18 carbon atoms, alkoxyof 2 to 18 carbon atoms, acyloxy of 2 to 18 carbon atoms in the acylmoiety, amino, acylamino, C^(*1) and C^(*2), independently from eachother being asymmetric carbons in configuration R, S, or in the racemicform RS, or a pharmaceutically acceptable salt or solvate thereof,optionally in conjugation or admixture with an inert non-toxicpharmaceutically acceptable diluent or carrier.
 2. A method according toclaim 1, wherein: X is selected from the following groups: carboxyl—COOH, dihydroxyphosphoryloxy —O—P(O)(OH)₂, hydroxysulfonyloxy—O—SO₂(OH), amino —NH₂, [amino(C₁-C₁₀)alkyl]aminocarbonyl—CONH(CH₂)_(n1)—NH₂, with n₁ being an integer from 1 to 10,[dicarboxy(C₁-C₅)alkyl]aminocarbonyl —CO—NH—CH(COOH)—(CH₂)_(n1)—COOH,with n₁ being an integer from 1 to 5,{carboxy[amino(C₁-C₅)alkyl]}aminocarbonyl—CO—NH—CH(COOH)—(CH₂)_(n1)—NH₂, with n₁ being an integer from 1 to 5,amino(C₁-C₁₅)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being an integerfrom 1 to 15, and Y is selected from the following groups:dihydroxyphosphoryloxy —O—P(O)(OH)₂, hydroxysulfonyloxy —SO₂(OH), amino—NH₂, hydroxyl —OH, amino(C₁-C₁₅alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, withn₁ being an integer from 1 to 15, dihydroxy (C₁-C₁₀) alkanoyloxy—O—CO—(CH₂)_(n1)—CHOH—CH₂OH, with n₁ being an integer from 1 to 10,hydroxy(C₁-C₁₀alkanoyloxy —O—CO—(CH₂)_(n1)—OH, with n₁ being an integerfrom 1 to 10, carboxy(C₁-C₅alkanoyloxy —O—CO—(CH₂)_(n1)—COOH, with n₁being an integer from 1 to 10, oxo(C₁-C₅)alkanoyloxy—O—CO—(CH₂)_(n1)—CHO, with n₁ being an integer from 1 to 5,[carboxy(C_(1-C) ₁₀)alkanoyl]amino(C₁-C₁₅)alkanoyloxy—O—CO—(CH₂)_(n1)—NH—CO—(CH₂)_(n2)—COOH, with n₁ being an integer from 1to 10, and n₂ being an integer from 1 to
 15. 3. A method according toclaim 1, wherein: X is selected from the following groups: carboxyl—COOH, dihydroxyphosphoryloxy —O—P(O)(OH)₂, hydroxysulfonyloxy—O—SO₂(OH), amino —NH₂, [amino(C₁-C₆)alkyl]aminocarbonyl—CONH(CH₂)_(n1)—NH₂, with n₁ being an integer from 1 to 6,[dicarboxy(C₁-C₅)alkyl]aminocarbonyl —CO—NH—CH(COOH)—(CH₂)_(n1)—COOH,with n₁ being an integer from 1 to 5,{carboxy[amino(C₁-C₅)alkyl]}aminocarbonyl —CO—NH—CH(COOH)—(CH₂)_(n1)—NH₂, with n₁ being an integer from 1 to 5,amino(C₂-C₁₂)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being an integerfrom 2 to 12, and Y is selected from the following groups:dihydroxyphosphoryloxy —O—P(O)(OH)₂, hydroxysulfonyloxy —O—SO₂(OH),amino —NH₂, hydroxyl —OH, amino(C₂-C₁₂)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂,with n₁ being an integer from 2 to 12, dihydroxy (C₃-C₇) alkanoyloxy—O—CO—(CH₂)_(n1)—CHOH—CH₂OH, with n₁ being an integer from 3 to 7,hydroxy(C₂-C₆)alkanoyloxy —O—CO—(CH₂)_(n1)—OH, with n₁ being an integerfrom 2 to 6, carboxy(C₃-C₆)alkanoyloxy —O—CO—(CH₂)_(n1)—COOH, with n₁being an integer from 3 to 6, oxo(C₂-C₅)alkanoyloxy—O—CO—(CH₂)_(n1)—CHO, with n₁ being an integer from 2 to 5,[carboxy(C₃-C₆)alkanoyl]amino(C₂-C₁₂)alkanoyloxy—O—CO—(CH₂)_(n1)—NH—CO—(CH₂)_(n2)—COOH, with n₁ being an integer from 3to 6, and n₂ being an integer from 2 to
 12. 4. A method according toclaim 1, wherein: X is selected from the following groups: —COOH,—O—P(O)(OH)₂, —O—SO₂(OH), —NH₂, —CO—NH—(CH₂)₃—NH₂, or —CONH—(CH₂)₆—NH₂,—CO—NH—CH(COOH)—CH₂—COOH, —CO—NH—CH(COOH)—(CH₂)₄—NH₂, —O—CO—(CH₂)₅—NH₂,and Y is selected from the following groups: —O—P(O)(OH)₂, —O—SO₂(OH),—NH₂, —OH, —O—CO—CH₂—NH₂ (2-aminoethanoyloxy), —O—CO—(CH₂)₂—NH₂(3-aminopropanoyloxy), —O—CO—(CH₂)₅—NH₂ (6-aminohexanoyloxy), or—O—CO—(CH₂)₁₁—NH₂ (12-aminododecanoyloxy), —O—CO—(CH₂)₄—CHOH—CH₂OH(6,7-dihydroxyheptanoyloxy), —O—CO—(CH₂)₅—OH (6-hydroxyhexanoyloxy),—O—CO—(CH₂)₂—COOH (3-carboxypropanoyloxy) —O—CO—(CH₂)₄—CHO(6-oxohexanoyloxy), —O—CO—(CH₂)₅—NH—CO—(CH₂)₂—COOH(3-carboxypropanoylamino hexanoyloxy).
 5. A method according to claim 1,wherein R₁ and R₂ are chosen among: —CO—CH₂—C*H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃, (3(C12O)C₁₄), —CO—CH₂—C*HOH(CH₂)₁₀—CH₃,(3 (HO)C₁₄), —CO—CH₃, (C2), —CO—CH₂—NH—CO—C*H[O—CO—(CH₂)₈—CH₃]—(CH₂)₅—CH₃, ([2(C₁₀O)C₈]NC₂),—CO—C*H[O—CO—(CH₂)₄—CH₃]—(CH₂)₅—CH₃, (2(C₆O)C8), —CO—(CH₂)₁₆—CH₃, (C18),—CO—CH₂—C*H[O—CH₂—C₆H₅]—(CH₂)₁₀—CH₃, (3(BnO)C₁₄), —CO—CH₂—C*H[O—P(O)(OH)₂]—(CH₂)₁₀—CH₃, (3 [(OH)₂—P(O)O]C₁₄), C* corresponding to anasymmetric carbon in configuration R, S, or in the racemic form RS, inthe formulae mentioned above.
 6. A method according to claim 1, wherein:R₁ is chosen among: —CO—CH₂—C^(**1)H [O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,(3(C₁₂O)C₁₄), —CO—CH₂—C^(**1)HOH—(CH₂)₁₀—CH₃, (3(HO)C₁₄), —CO—CH₃, (C2),—CO—CH₂—NH—CO—C^(**1)H[O—CO—(CH₂)₈]—(CH₂)₅—CH₃, ([2 (C₁₀O)C₈]NC₂),—CO—C^(**1)H [O—CO—(CH₂)₄—CH₃]—(CH₂)₅—CH₃, (2 (C₆O)C₈), —CO—(CH₂)₁₆—CH₃,(C18), R₂ is chosen among:—CO—CH₂—C^(**2)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃, (3 (C₁₂O)C₁₄),—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, (3 (HO)C₁₄),—CO—CH₂—C^(**2)H[O—CH₂—C₆H₅]—(CH₂)₁₀—CH₃, (3 (BnO)C₁₄),—CO—CH₂—C^(**2)H[O—P(O)(OH)₂]—(CH₂)₁₀—CH₃, (3[(OH)₂—P(O)O]C₁₄). C^(**1)and C^(**2) corresponding to asymmetric carbons in configuration R, S,or in the racemic form RS, in the formulae mentioned above.
 7. A methodaccording to claim 1, wherein the administered compound has the generalformula (II):

Wherein X, Y, R₁, R₂, and m are as defined in anyone of claims 1 to 6,C^(*1) is in configuration R, S, or is the racemic RS, and C^(*2) is inconfiguration R.
 8. A method according to claim 1, wherein theadministered compound is selected among those of formula (I) wherein:X=—O—P(O)(OH)₂, Y=—O—P(O)(OH)₂,R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,R₂=—CO—CH₂—C^(**2)HOH(CH₂)₁₀—CH₃, m=2, C^(*1) is in configuration S,C^(*2) is in configuration R, C^(**1) and C^(**2) are in configurationR, i.e.(3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol (1,10)-bis-dihydrogenophosphate (OM-294-DP(S,R)), X=—O—P(O)(OH)₂, Y=—O—CO—(CH₂)₄—CHOH—CH₂OH,R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=2, C^(*1) is in configuration S,and C^(*2) is in configuration R, C^(**1) and C^(**2) are inconfiguration R, i.e. (3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoyl amino]-decan-1, 10-diol 1-dihydrogenophosphate10(6,7-dihydroxyheptanoate) (OM-197-MP-HD (S,R)), X=—COOH,Y=—O—CO—(CH₂)₄—CHOH—CH₂OH,R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=1, C^(*1) is in configuration S,and C^(*2) is in configuration R, C^(**1) and C^(**2) are inconfiguration R, i.e. N—[(R)-3-dodecanoyloxytetradecanoyl]-L-asparticacid, α-N-{(4R)-5-hydroxy-4-[(R)-3-hydroxytetradecanoylamino]pentyl}amide5-O-(6,7-dihydroxyheptanoate) (OM-197-MC-HD (S,R)), X=—O—P(O)(OH)₂,Y=—O—CO—(CH₂)₅—NH₂, R₁=—CO—CH₂—C^(**1)H [O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=2, C^(*1) is in configuration S,and C^(*2) is in configuration R, C^(**1) and C^(**2) are inconfiguration R, i.e. (3S,9R)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1, 10-diol 1-dihydrogeno phosphate10-(6-aminohexanoate) (OM-197-MP-AC (S,R)), X=—NH₂, Y=—O—P(O)(OH)₂,R₁=—CO—CH₂—C^(**1)H[O—CO—(CH₂)₁₀—CH₃]—(CH₂)₁₀—CH₃,R₂=—CO—CH₂—C^(**2)HOH—(CH₂)₁₀—CH₃, m=4, C^(*1) is in configuration S,and C^(*2) is in configuration R, C^(**1) and C^(**2) are inconfiguration R, i.e.(5S,11R)-1-Amino-5-[(R)-3-dodecanoyloxytetradecanoylamino]-6-oxo-7-aza-11-[(R)-3-hydroxytetradecanoylamino]-dodecan-12-ol 12-dihydrogen phosphate (OM-294-BA-MP(S,R).
 9. A method according to claim 1, wherein said disease ordisorder is related to an overproduction of inflammatory cytokines byactivated T lymphocytes, monocytes, or antigen presenting cells in theorganism, wherein the inflammatory cytokines or inflammatory markersbelong to the group consisting of IL-4, IL-5 and IL-13.
 10. A methodaccording to claim 1 which consists in administering a therapeuticallyeffective amount of said compound in a pharmaceutically-acceptablecarrier, excipient or formulation, via a mucosal or parenteral route.11. A method according to claim 1 which consists in administering atherapeutically effective amount of said compound preferentially via theperitoneal, subcutaneous, oral, intranasal, sublingual, or aerosolroutes.
 12. A method according to claim 1, wherein the dosages of theimmunomodulatory compound range from 0.01 to 50 mg/m² in humans.
 13. Amethod according to claim 11, wherein X is selected from the followinggroups: carboxyl —COOH, dihydroxyphosphoryloxy —O—P(O)(OH)₂,hydroxysulfonyloxy —O—SO₂(OH), amino —NH₂,[amino(C₁-C₆)alkyl]aminocarbonyl —CONH(CH₂)_(n1)—NH₂, with n₁ being aninteger from 1 to 6, [dicarboxy(C₁-C₅)alkyl]aminocarbonyl—CO—NH—CH(COOH)—(CH₂)_(n1)—COOH, with n₁ being an integer from 1 to 5,{carboxy[amino(C₁-C₅)alkyl]}aminocarbonyl —CO—NH—CH(COOH)—(CH₂)_(n1)—NH₂with n₁ being an integer from 1 to 5, amino(C₂-C₁₂)alkanoyloxy—O—CO—(CH₂)_(n1)—NH₂, with n₁ being an integer from 2 to 12, and Y isselected from the following groups: dihydroxyphosphoryloxy —O—P(O)(OH)₂,hydroxysulfonyloxy —O—SO₂ (OH), amino —NH₂, hydroxyl —OH,amino(C₂-C₁₂)alkanoyloxy —O—CO—(CH₂)_(n1)—NH₂, with n₁ being an integerfrom 2 to 12, dihydroxy(C₃-C₇)alkanoyloxy —O—CO—(CH₂)_(n1)—CHOH—CH₂OH,with n₁ being an integer from 3 to 7, hydroxy(C₂-C₆)alkanoyloxy—O—CO—(CH₂)_(n1)—OH, with n₁ being an integer from 2 to 6,carboxy(C₃-C₆)alkanoyloxy —O—CO—(CH₂)_(n1)—COOH, with n₁ being aninteger from 3 to 6, oxo(C₂-C₅)alkanoyloxy —O—CO—(CH₂)_(n1)—CHO, with n₁being an integer from 2 to 5,[carboxy(C₃-C₆)alkanoyl]amino(C₂-C₁₂)alkanoyloxy—O—CO—(CH₂)_(n1)—NH—CO—(CH₂)_(n2)—COOH, with n₁ being an integer from 3to 6, and n₂ being an integer from 2 to 12.